Methods and compositions for multiplex pcr

ABSTRACT

The present invention provides methods, compositions, kits, systems and apparatus that are useful for multiplex PCR of one or more nucleic acids present in a sample. In particular, various target-specific primers are provided that allow for the selective amplification of one or more target sequences. In one aspect, the invention relates to target-specific primers useful for the selective amplification of one or more target sequences associated with cancer or inherited disease. In some aspects, amplified target sequences obtained using the disclosed methods, kits, systems and apparatuses can be used in various downstream processes including nucleic acid sequencing and used to detect the presence of genetic variants.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority under 35 U.S.C. §119(e) toU.S. Provisional Application No. 61/479,952, filed Apr. 28, 2011, U.S.Provisional Application No. 61/531,583, filed Sep. 6, 2011, U.S.Provisional Application No. 61/531,574, filed Sep. 6, 2011, U.S.Provisional Application No. 61/538,079, filed Sep. 22, 2011, U.S.Provisional Application No. 61/564,763, filed Nov. 29, 2011, U.S.Provisional Application No. 61/578,192, filed Dec. 20, 2011, U.S.Provisional Application No. 61/594,160 filed Feb. 2, 2012, U.S.Provisional Application No. 61/598,881 filed Feb. 14, 2012, U.S.Provisional Application No. 61/598,892 filed Feb. 14, 2012, U.S.Provisional Application No. 61/625,596 filed Apr. 17, 2012, and U.S.Provisional Application No. 61/639,017 filed Apr. 26, 2012 entitled“METHODS AND COMPOSITIONS FOR MULTIPLEX PCR”, the disclosures of whichare incorporated herein by reference in their entireties.

SEQUENCE LISTING

This application hereby incorporates by reference the material of theelectronic Sequence Listing filed concurrently herewith. The material inthe electronic Sequence Listing is submitted as a text (.txt) fileentitled “2012_(—)04_(—)25 LT00503 US_ST25.txt” created on Apr. 25,2012, which has a file size of 18943 KB, and is herein incorporated byreference in its entirety.

TECHNICAL FIELD

In some embodiments, the disclosure relates generally to methods,compositions, systems, apparatuses and kits for amplifying one or moretarget sequences within a sample containing a plurality of targetsequences. Optionally, a plurality of target sequences, for example atleast 10, 50, 100, 500, 1000, 2500, 5000, 7500, 10000, 25000, 50000 or100000, are amplified within a single amplification reaction. In someembodiments, the disclosure relates generally to methods, compositions,systems, apparatuses and kits for amplifying one or more targetsequences from a single source, such as genomic DNA or formalin-fixedparaffin-embedded (FFPE) DNA. In particular, methods, kits, systemsapparatuses and compositions useful for amplifying one or more targetsequences using primers having a cleavable group are disclosed.

BACKGROUND

Several biological applications involve the selective amplification ofnucleic acid molecules within a population. For example, next-generationsequencing methods can involve the analysis of selected targets within alarge population of nucleic acid molecules. For such applications, itcan be useful to increase the total number of targets that can beselectively amplified from a population within a single amplificationreaction. Such selective amplification is typically achieved through useof one or more primers that can selectively hybridize to, or selectivelypromote the amplification of, a particular target nucleic acid molecule.Such selective amplification can be complicated by the formation ofamplification artifacts, such as primer-dimers and the like. Theformation of such amplification artifacts (also referred to herein asnonspecific amplification products) can consume critical amplificationreagents, e.g., nucleotides, polymerase, primers, etc. Furthermore, suchartifacts can frequently have shorter length relative to the intendedproduct and in such situations can amplify more efficiently than theintended products and dominate the reaction output. Selectiveamplification can also be complicated by the formation of‘superamplicons’, i.e., the formation of a extended amplicon, which canoccur when extension of a first primer is extended through an adjacenttarget nucleic acid sequence, thereby creating a long non-specificamplification product, which can act as a template for extension with asecond primer. The formation of such artifacts in amplificationreactions, even when only a single pair of primers is employed, cancomplicate downstream applications such as qPCR, cloning, geneexpression analysis and sample preparation for next-generationsequencing. In some downstream applications, including severalnext-generation sequencing methods, this problem can be compounded bythe requirement to practice a secondary amplification step, since theartifacts can be further amplified during the secondary amplification.For example, downstream sequencing applications can involve thegeneration of clonally amplified nucleic acid populations that areindividually attached to separate supports, such as beads, usingemulsion PCR (“emPCR”) and enrichment for clonal amplicons performed viapositive selection. In such applications, the artifacts can be carriedall the way through the library generation process to the emPCR stage,producing DNA capture beads that include non-specific amplificationproducts. These artifact-containing beads can be selected for during theenrichment process with the template containing beads but aregenetically non-informative.

Nucleic acid molecules amplified in a multiplex PCR reaction can be usedin many downstream analysis or assays with, or without, furtherpurification or manipulation. For example, the products of a multiplexPCR reaction (amplicons) when obtained in sufficient yield can be usedfor single nucleotide polymorphism (SNP) analysis, genotyping, copynumber variation analysis, epigenetic analysis, gene expressionanalysis, hybridization arrays, analysis of gene mutations including butnot limited to detection, prognosis and/or diagnosis of disease states,detection and analysis of rare or low frequency allele mutations,nucleic acid sequencing including but not limited to de novo sequencingor targeted resequencing, and the like.

Exemplary next-generation sequencing systems include the Ion TorrentPGM™ sequencer (Life Technologies) and the Ion Torrent Proton™ Sequencer(Life Technologies), which are ion-based sequencing systems thatsequence nucleic acid templates by detecting ions produced as abyproduct of nucleotide incorporation. Typically, hydrogen ions arereleased as byproducts of nucleotide incorporations occurring duringtemplate-dependent nucleic acid synthesis by a polymerase. The IonTorrent PGM™ sequencer and Ion Proton™ Sequencer detect the nucleotideincorporations by detecting the hydrogen ion byproducts of thenucleotide incorporations. The Ion Torrent PGM™ sequencer and IonTorrent Proton™ sequencer include a plurality of nucleic acid templatesto be sequenced, each template disposed within a respective sequencingreaction well in an array. The wells of the array are each coupled to atleast one ion sensor that can detect the release of H⁺ ions or changesin solution pH produced as a byproduct of nucleotide incorporation. Theion sensor comprises a field effect transistor (FET) coupled to anion-sensitive detection layer that can sense the presence of H⁺ ions orchanges in solution pH. The ion sensor provides output signalsindicative of nucleotide incorporation which can be represented asvoltage changes whose magnitude correlates with the H⁺ ion concentrationin a respective well or reaction chamber. Different nucleotide types areflowed serially into the reaction chamber, and are incorporated by thepolymerase into an extending primer (or polymerization site) in an orderdetermined by the sequence of the template. Each nucleotideincorporation is accompanied by the release of H⁺ ions in the reactionwell, along with a concomitant change in the localized pH. The releaseof H⁺ ions is registered by the FET of the sensor, which producessignals indicating the occurrence of the nucleotide incorporation.Nucleotides that are not incorporated during a particular nucleotideflow will not produce signals. The amplitude of the signals from the FETmay also be correlated with the number of nucleotides of a particulartype incorporated into the extending nucleic acid molecule therebypermitting homopolymer regions to be resolved. Thus, during a run of thesequencer multiple nucleotide flows into the reaction chamber along withincorporation monitoring across a multiplicity of wells or reactionchambers permit the instrument to resolve the sequence of many nucleicacid templates simultaneously. Further details regarding thecompositions, design and operation of the Ion Torrent PGM™ sequencer canbe found, for example, in U.S. patent application Ser. No. 12/002,781,now published as U.S. Patent Publication No. 2009/0026082; U.S. patentapplication Ser. No. 12/474,897, now published as U.S. PatentPublication No. 2010/0137143; and U.S. patent application Ser. No.12/492,844, now published as U.S. Patent Publication No. 2010/0282617,all of which applications are incorporated by reference herein in theirentireties. In some embodiments, amplicons can be manipulated oramplified through bridge amplification or emPCR to generate a pluralityof clonal templates that are suitable for a variety of downstreamprocesses including nucleic acid sequencing. In one embodiment, nucleicacid templates to be sequenced using the Ion Torrent PGM™ or Ion TorrentProton™ system can be prepared from a population of nucleic acidmolecules using one or more of the target-specific amplificationtechniques outlined herein. Optionally, following target-specificamplification a secondary and/or tertiary amplification processincluding, but not limited to a library amplification step and/or aclonal amplification step such as emPCR can be performed.

As the number of nucleic acid targets desired to be amplified within asample nucleic acid population increases, the challenge of selectivelyamplifying these targets while avoiding the formation of undesirableamplification artifacts can correspondingly increase. For example, theformation of artifacts including primer-dimers and superamplicons can bea greater issue in multiplex PCR reactions where PCR primer pairs formultiple targets are combined in a single reaction tube andco-amplified. In multiplex PCR, the presence of additional primer pairsat elevated concentrations relative to the template DNA makesprimer-primer interactions, and the formation of primer-dimers and otherartifacts, more likely.

Current methods for avoiding or reducing the formation of artifacts,such as primer-dimers, during nucleic acid amplification center aroundthe primer design process and often utilize dedicated software packages(e.g., DNAsoftwares's Visual OMP, MultiPLX, ABI's Primer Express, etc.)to design primer pairs that are predicted to exhibit minimal interactionbetween the other primers in the pool during amplification. Through theuse of such software, primers can be designed to be as target-specificor amplicon-specific as possible, and often are grouped into subsets tominimize primer-primer interactions, primer-dimer formation andsuperamplicons. Stringent design parameters, however, limit the numberof amplicons that can be co-amplified simultaneously and in some casesmay prevent the amplification of some amplicons altogether. Othercurrent methods require the use of multiple PCR primer pools tosegregate primers into non-overlapping pools to minimize or preventprimer artifacts during the amplification step. Other methods includethe use of multiple primer pools or single plex reactions to enhance theoverall yield of amplification product per reaction. In a multiplex PCRreaction, each primer pair competes in the amplification reaction withadditional primer pairs for a finite amount of dNTPs, polymerase andother reagents. There is therefore a need for improved methods,compositions, systems, apparatuses and kits that allow for the selectiveamplification of multiple target nucleic acid molecules within apopulation of nucleic acid molecules while avoiding, or minimizing, theformation of artifacts (also referred to as non-specific amplificationproducts), including primer dimers. There is also a need for improvedmethods, compositions, systems, apparatuses and kits that allow for theselective amplification of multiple target nucleic acid molecules from asingle nucleic acid sample, such as genomic DNA and/or formalin-fixedparaffin embedded (FFPE) DNA while avoiding, or minimizing, theformation of artifacts. There is also a need in the art for improvedmethods, compositions, systems and kits that allow for the simultaneousamplification of thousands of target-specific nucleic acid molecules ina single reaction, which can be used in any applicable downstream assayor analysis.

The practice of the present subject matter may employ, unless otherwiseindicated, conventional techniques and descriptions of organicchemistry, molecular biology (including recombinant techniques), cellbiology, and biochemistry, which are within the skill of the art. Suchconventional techniques include, but are not limited to, preparation ofsynthetic polynucleotides, polymerization techniques, chemical andphysical analysis of polymer particles, preparation of nucleic acidlibraries, nucleic acid sequencing and analysis, and the like. Specificillustrations of suitable techniques can be used by reference to theexamples provided herein. Other equivalent conventional procedures canalso be used. Such conventional techniques and descriptions can be foundin standard laboratory manuals such as Genome Analysis: A LaboratoryManual Series (Vols. I-IV), PCR Primer: A Laboratory Manual, andMolecular Cloning: A Laboratory Manual (all from Cold Spring HarborLaboratory Press), Hermanson, Bioconjugate Techniques, Second Edition(Academic Press, 2008); Merkus, Particle Size Measurements (Springer,2009); Rubinstein and Colby, Polymer Physics (Oxford University Press,2003); and the like.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which these inventions belong. All patents, patentapplications, published applications, treatises and other publicationsreferred to herein, both supra and infra, are incorporated by referencein their entirety. If a definition and/or description is set forthherein that is contrary to or otherwise inconsistent with any definitionset forth in the patents, patent applications, published applications,and other publications that are herein incorporated by reference, thedefinition and/or description set forth herein prevails over thedefinition that is incorporated by reference.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having” or any other variation thereof, areintended to cover a non-exclusive inclusion. For example, a process,method, article, or apparatus that comprises a list of features is notnecessarily limited only to those features but may include otherfeatures not expressly listed or inherent to such process, method,article, or apparatus. Further, unless expressly stated to the contrary,“or” refers to an inclusive-or and not to an exclusive-or. For example,a condition A or B is satisfied by any one of the following: A is true(or present) and B is false (or not present), A is false (or notpresent) and B is true (or present), and both A and B are true (orpresent).

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and form a partof the specification, illustrate one or more exemplary embodiments andserve to explain the principles of various exemplary embodiments. Thedrawings are exemplary and explanatory only and are not to be construedas limiting or restrictive in any way.

FIG. 1A-FIG. 1E₂ are schematic outlining an exemplary embodiment of amethod utilizing degradable amplification primers according to thedisclosure.

FIG. 2 is a schematic outlining an exemplary embodiment of a methodobtaining a target-specific amplicon library according to thedisclosure.

FIG. 3A-FIG. 3B show examples of elution profiles for exemplaryunmodified and modified primer pools. FIG. 3A shows a significant andpredominant production of primer-dimers when using an exemplary set ofstandard multiplex primers. FIG. 3B shows a decrease in primer-dimersand an overall increase in expected amplicon product (104 bp) when usingan exemplary set of modified multiplex primers as exemplified by theapplication.

FIG. 4A-FIG. 4H show the effect of increasing amplicon GC content inboth an exemplary 94-plex and an exemplary 380-plex reaction.

FIG. 5 shows quantification of the abundance and reproducibility of anexemplary multiplex reaction on genomic DNA using primer set HSMv12. Thedata is averaged across individual 3 runs performed on an Ion TorrentPGM™ Sequencer (Life Technologies). The level of coverage per ampliconis provided as Log of Counts.

FIG. 6A-6B show quantification of the abundance and reproducibility ofan exemplary multiplex reaction on genomic DNA when analyzed using anIon Torrent PGM™ Sequencer (Life Technologies). The data shows thenumber of reads per amplicon for both the forward (FIG. 6A) and reverseprimers (FIG. 6B) in the primer pool.

FIG. 7 shows quantification of the abundance and reproducibility of anexemplary 384-plex reaction on a genomic DNA sample across 7 individualruns on an Ion Torrent PGM™ Sequencer (Life Technologies). The averagenumber of reads per amplicon is 400.

FIG. 8 shows quantification of the abundance and reproducibility of anexemplary 411-plex PCR of genomic DNA across 7 individual runs on an IonTorrent PGM™ Sequencer (Life Technologies). The average number of readsper amplicon is 400.

FIG. 9 shows an image of agarose gel electrophoresis visualizingexemplary amplification products (lanes 2 and 4) after performing amultiplex PCR and library amplification on an FFPE sample according toan exemplary embodiment.

FIG. 10 shows quantification of the abundance and reproducibility ofdata obtained from an FFPE DNA sample (10 ng) in an exemplary 384-plexPCR obtained using an Ion Torrent PGM™ Sequencer (Life Technologies).The average number of reads per amplicon is 400.

FIG. 11A-11B show quantification of the abundance and reproducibility ofdata obtained from a FFPE DNA sample (10 ng) after an exemplary 94-plexreaction. The data shows the number of reads per amplicon for both theforward (FIG. 11A) and reverse primers (FIG. 11B) in the primer pool.

FIG. 12 shows a graph reporting the detection of mutations in codon 12and codon 13 of the KRAS gene obtained by performing multiplex PCR andlibrary amplification of control DNA and sequencing on an Ion TorrentPGM™ Sequencer (Life Technologies).

FIG. 13 (Part 1)-FIG. 13 (Part 3) show sequencing alignment dataidentifying 6 mutations of the cystic fibrosis (CFTR) gene in a samplewhen using exemplary modified multiplex primers and exemplary libraryamplification process according to the disclosure.

FIG. 14 shows the frequencies of twelve different amplicons obtained fora region of the CLTCL1 gene in several DNA samples containing known copynumber variation. The amplicons were obtained according to exemplaryembodiments of the disclosure.

FIG. 15 shows the frequencies of four different amplicons obtained for aregion of the IKZF1 gene in several DNA samples containing known copynumber variation. The amplicons were obtained according to exemplaryembodiments of the multiplex PCR methods disclosed herein.

FIG. 17 illustrates a system for designing primers or assays accordingto an exemplary embodiment.

FIG. 18 illustrates a system for designing primers or assays accordingto an exemplary embodiment.

FIG. 19 illustrates an amplicon sequence including an insert sequencesurrounded by a pair of primers designed according to an exemplaryembodiment.

FIG. 20 illustrates PCR amplification of an amplicon sequence (which maybe referred to as “tile” herein) including an insert surrounded by apair of primers designed according to an exemplary embodiment.

FIG. 21 (Part 1)-FIG. 21 (Part 3) illustrate a set of candidateamplicons for a given target region, each including an insert surroundedby a pair of primers, for tiling and pooling according to an exemplaryembodiment.

FIG. 22 illustrates a method according to an exemplary embodiment.

FIG. 23 illustrates a method for tiling a plurality of amplicons for oneor more given targets according to an exemplary embodiment.

FIG. 24 illustrates a method for determining tiles for one or more giventargets and candidate amplicons according to an exemplary embodiment.

FIG. 25A illustrates a set of candidate amplicons for covering a giventarget region, each including an insert surrounded by a pair of primers,for tiling and pooling according to an exemplary embodiment.

FIG. 25B illustrates a set of vertices for generating a graph accordingto an exemplary embodiment.

FIG. 26A illustrates the 15 candidate amplicons of FIG. 25A, except thatthree “initial” amplicons having at least some overlap between theirinsert and the beginning of the target region are highlighted.

FIG. 26B illustrates the connection of a source vertex to three verticescorresponding to the initial amplicons of FIG. 26A with edges.

FIG. 27A illustrates the 15 candidate amplicons of FIG. 25A, except thatthree “terminal” amplicons having at least some overlap between theirinsert and the end of the target region are highlighted.

FIG. 27B illustrates the connection of a sink vertex to three verticescorresponding to the terminal amplicons of FIG. 27A with edges.

FIG. 28A illustrates the 15 candidate amplicons of FIG. 25A, except thatvarious amplicons for building internal edges are highlighted.

FIG. 28B illustrates the connection of some amplicon insert vertices tosubsequent, proper, overlaps according to an exemplary embodiment.

FIG. 29A illustrates the connection of additional amplicon insertvertices to subsequent, proper, overlaps according to an exemplaryembodiment.

FIG. 29B illustrates the 15 candidate amplicons of FIG. 25A, along withthe basis for the gap shown in FIG. 29A according to an exemplaryembodiment.

FIG. 30A illustrates three possible additional edges that could be usedfrom source to sink to tile the target in this example according to anexemplary embodiment.

FIG. 30B illustrates an exemplary definition of an edge cost functionassigning a cost to each one of the graph's edges linking ampliconvertices according to an exemplary embodiment.

FIG. 30C illustrates the least-cost path from source to sink in theexample of FIG. 30B according to an exemplary embodiment.

FIG. 31 illustrates the 15 candidate amplicons of FIG. 25A, except thatthe five amplicons corresponding to the vertices forming the least-costpath shown in FIG. 30C are highlighted.

FIG. 32 illustrates three amplicons assigned to a first pool and twoamplicons assigned to a second pool according to an exemplaryembodiment.

FIG. 33A illustrates a minimal distance between amplicons according toan exemplary embodiment.

FIGS. 33B-D illustrate several problems, including primer “racecondition,” preferential amplification of sub amplicons, and superamplicons that may be ameliorated by using a minimal distance asillustrated in FIG. 33A.

FIG. 34 illustrates a method for pooling amplicons across a plurality ofpools according to an exemplary embodiment.

FIG. 35 illustrates a method according to an exemplary embodiment.

SUMMARY

In some embodiments, the disclosure relates generally to methods,compositions, systems, apparatuses and kits for performing multiplexamplification of nucleic acids. In some embodiments, the method includesamplifying a plurality of target sequences within a sample including twoor more target sequences. Optionally, multiple target sequences ofinterest from a sample can be amplified using one or moretarget-specific primers in the presence of a polymerase underamplification conditions to produce a plurality of amplified targetsequences. The amplifying optionally includes contacting a nucleic acidmolecule including at least one target sequence with one or moretarget-specific primers and at least one polymerase under amplificationconditions. The contacting can produce one or more amplified targetsequences.

In some embodiments, the disclosed methods (and related compositions,systems, apparatuses and kits) can include ligating at least one adapterto at least one of the amplified target sequences to produce one or moreadapter-ligated amplified target sequences. The adapter can include atleast one sequence that is substantially non-complementary to the targetsequence, to the amplified target sequence, and/or to the nucleic acidmolecule.

In some embodiments, the amplifying can produce least two amplifiedtarget sequences that are less than 50% complementary to each other. Insome embodiments, at least one amplified target sequence issubstantially non-complementary to another target sequence in thesample. In some embodiments, an amplified target sequence can besubstantially noncomplementary to any one or more nucleic acid moleculesin the sample that does not include the target sequence.

In some embodiments, the disclosed methods (as well as relatedcompositions, systems, apparatuses and kits) can involve reamplifying atleast one of the amplified target sequences c. For example, anadapter-ligated amplified target sequence can be reamplified to produceat least one reamplified adapter-ligated amplified target sequence. Insome embodiments, at least one of the adapter-ligated amplified targetsequences can be contacted with one or more adapters or theircomplement, and a polymerase under amplification conditions to produceat least one reamplified adapter-ligated amplified target sequence. Insome embodiments, at least one adapter or its complement issubstantially non-complementary to at least one amplified targetsequence.

In some embodiments, the disclosure relates generally to compositions(as well as related methods, kits, apparatuses and systems using suchcompositions) comprising one or more target-specific primers useful forhybridizing to, and optionally amplifying, at least one target sequencein a sample. In some embodiments, the composition can include aplurality of target-specific primers useful for amplifying one, two ormore target sequences in a sample. The compositions can further includeone or more adapters.

In some embodiments, a target sequence includes one or more mutationalhotspots, single nucleotide polymorphisms (SNPs), short tandem repeats(STRs), coding regions, exons and genes. In some embodiments, the numberof target sequences amplified by one or more of the methods using thecompositions (and related kits, apparatuses and systems) disclosedherein can be dozens, hundreds or thousands of target sequences in asingle reaction. In some embodiments, the number of different targetsamplified in a single multiplex amplification can be at least 100, 300,500, 750, 1000, 2500, 5000, 7500, 10000, 12500, 15000 or greater.

In some embodiments, a target-specific primer, adapter, amplified targetsequence or nucleic acid molecule can include one or more cleavablemoieties, also referred to herein as cleavable groups. Optionally, themethods can further include cleaving at least one cleavable group of thetarget-specific primer, adapter, amplified target sequence or nucleicacid molecule. The cleaving can be performed before or after any of theother steps of the disclosed methods. In some embodiments, the cleavagestep occurs after the amplifying and prior to the ligating. In oneembodiment, the cleaving includes cleaving at least one amplified targetsequence prior to the ligating. The cleavable moiety can be present in amodified nucleotide, nucleoside or nucleobase. In some embodiments, thecleavable moiety can include a nucleobase not naturally occurring in thetarget sequence of interest. In some embodiments, uracil or uridine canbe incorporated into a DNA-based nucleic acid as a cleavable group. Inone exemplary embodiment, a uracil DNA glycosylase can be used to cleavethe cleavable group from the nucleic acid. In another embodiment,inosine can be incorporated into a DNA-based nucleic acid as a cleavablegroup. In one exemplary embodiment, EndoV can be used to cleave near theinosine residue and a further enzyme such as Klenow can be used tocreate blunt-ended fragments capable of blunt-ended ligation. In anotherexemplary embodiment, the enzyme hAAG can be used to cleave inosineresidues from a nucleic acid creating abasic sites that can be furtherprocessed by one or more enzymes such as Klenow to create blunt-endedfragments capable of blunt-ended ligation.

In some embodiments, the methods disclosed herein (as well as relatedkits, compositions, apparatuses and systems) can include amplifying atleast two target sequences of the sample (e.g., a first target sequenceand a second target sequence) that are different from each other. Insome embodiments, the methods disclosed herein (as well as related kits,compositions, apparatuses and systems) include simultaneously amplifyinga first target sequence and a second target sequence that are less than50% complementary to each other. In some embodiments, the first targetsequence and a second target sequence are substantiallynon-complementary to each other.

In some embodiments, the methods disclosed herein (as well as relatedkits, compositions, apparatuses and systems) can include amplifyingusing at least two target-specific primers (e.g., a firsttarget-specific primer and a second target-specific primer) that aredifferent from each other. In some embodiments, a first target-specificprimer can be at least 50% complementary to at least some portion of afirst target sequence. In some embodiments, the first target-specificprimer can be substantially noncomplementary to another target sequencein the sample. For example, the first target-specific primer can besubstantially noncomplementary to a second target sequence. Optionally,the first target-specific primer can be substantially complementary to afirst target sequence within a sample and can be substantiallynoncomplementary to any portion of any other nucleic acid moleculewithin the sample other than the first target sequence.

Optionally, methods of multiplex amplification disclosed herein includeamplifying at least a portion of a target sequence in a sample using atleast one target-specific primer that is substantially complementary toat least some portion of a nucleic acid molecule that includes acorresponding target sequence. In some embodiments, the at least onetarget-specific primer is substantially complementary to at least someportion of the corresponding target sequence. In some embodiments, theamplifying can include using a primer pair including a target-specificforward primer and a target-specific reverse primer. In someembodiments, the target-specific primer can include at least onesequence that is substantially complementary or substantially identicalto at least some portion of a nucleic acid molecule that includes thecorresponding target sequence or its complement. Optionally, thetarget-specific primer is not substantially complementary to any othernucleic acid molecule present in the sample. In some embodiments, thetarget-specific primer can include at least one sequence that issubstantially complementary or substantially identical to at least someportion of a corresponding target sequence or its complement. In someembodiments, the target-specific primer can include at least onesequence that is complementary or identical to at least some portion ofa corresponding target sequence or its complement. In some embodiments,a target-specific primer does not include any nucleic acid sequence thatis at least 5 contiguous nucleotides, 8 nucleotides, 10 contiguousnucleotides, or 15 contiguous nucleotides in length, and that issubstantially noncomplementary to at least some portion of itscorresponding target sequence. In some embodiments, a target-specificprimer can hybridize under stringent conditions to at least some portionof a corresponding target sequence in the sample. In some embodiments,at least one of the target-specific primers is not substantiallycomplementary to any nucleic acid sequence present in the sample otherthan its corresponding target sequence.

In some embodiments, one or more target-specific primers can be designedto exclude one or more sequence motifs. For example, at least one of thetarget-specific primers may be designed to not include a tripletnucleotide motif that is repeated 5 or more times in the target-specificprimer. Optionally, at least one of the target-specific primers may bedesigned to not include the nucleotide sequence “ACA”, repeated 3 ormore times. Further, at least one of the target-specific primers may bedesigned to not include a homopolymer greater than 8 nucleotides inlength. Optionally, at least one of the target-specific primers of themethods disclosed herein may be designed to possess a GC content of lessthan 85%.

In some embodiments, one or more of the methods of amplifying disclosedherein includes performing a target-specific amplification. Performingthe target-specific amplification can include amplifying one or moretarget sequences using one or more exclusively target-specific primers,i.e., primers that do not include any shared or universal sequencemotifs. Typically, one or more of the target-specific primers aresubstantially complementary to at least some portion of theircorresponding target sequence, or to some portion of the nucleic acidmolecule including the corresponding target sequence. In someembodiments, one, some or all of the target-specific primers aresubstantially complementary to at least some portion of theircorresponding target sequence, or to some portion of the nucleic acidmolecule including the corresponding target sequence, across their(i.e., the primers') entire length.

In some embodiments, a nucleic acid molecule in a sample, an amplifiedtarget sequence, an adapter or a target-specific primer includes a 5′end and a 3′ end. The 5′ end can include a free 5′ phosphate group orits equivalent; the 3′ end can include a free 3′ hydroxyl group or itsequivalent. Optionally, the ends of an amplified target sequence can besubstantially non-complementary to the ends of another amplified targetsequence. In some embodiments, the 3′ end can include about 30nucleotides, or about 15 nucleotides from the 3′ hydroxyl group. In someembodiments, the 5′ end can include about 30 nucleotides, or about 15nucleotides, from the 5′ phosphate group. In some embodiments, any oneamplified target sequence having a 3′ end and 5′ end can besubstantially non-complementary to any portion of any other amplifiedtarget sequence.

Optionally, the disclosed methods can further include ligating one ormore adapters including a universal priming sequence to the amplifiedproduct formed as a result of such target-specific amplification. Forexample, in some embodiments, one or more adapters can be ligated to anamplified target sequence. Optionally, an adapter that is ligated to anamplified target sequence is susceptible to exonuclease digestion. Insome embodiments, an adapter susceptible to exonuclease digestion can beligated to the 3′ end of an amplified target sequence. In someembodiments, an adapter ligated to an amplified target sequence does notinclude a protecting group. In some embodiments, the adapter does notinclude a protecting group that can prevent nucleic acid degradation ordigestion under degrading or digesting conditions. Subsequent enzymaticdigestion of the adapter-ligated amplified target sequence in thepresence of nucleic acids that do not include a protecting group, offersa means for selective digestion of the unprotected nucleic acids. Insome embodiments, an adapter can include a DNA barcode or taggingsequence.

In some embodiments, the methods disclosed herein (as well as relatedkits, systems, apparatuses and compositions) can include contacting anamplified target sequence having a 3′ end and a 5′ end with a ligationreaction mixture. In some embodiments, a ligation reaction mixture caninclude one or more adapters and a ligase to produce at least oneadapter-ligated amplified target sequence. In some embodiments, none ofthe adapters in a ligation mixture, prior to the ligating, includes atarget-specific sequence. In some embodiments, none of the adapters inthe ligation mixture, prior to ligating, includes a sequence that issubstantially complementary to a 3′ end or a 5′ end of an amplifiedtarget sequence. Optionally, the 3′ end or the 5′ end of an amplifiedtarget sequence includes about 30 nucleotides, and in some instancesrefers to about 15 nucleotides from the 3′ end or the 5′ end of anamplified target sequence. In some embodiments, none of the adapters ina ligation mixture, prior to ligating, can hybridize under highstringency, to some portion of an amplified target sequence. In someembodiments, ligating can include direct ligation of one or moreadapters to one or more amplified target sequences. In one embodiment,ligating can include performing a blunt-ended ligation. For example, theprocess of blunt-ended ligation can include ligating a substantiallyblunt-end double-stranded amplified target sequence to a substantiallyblunt-ended double-stranded adapter. In some embodiments, ligating doesnot include one or more additional oligonucleotide adapters prior toligating an adapter to an amplified target sequence.

In some embodiments, the disclosure relates generally to methods forperforming amplification of a target sequence (as well as relatedcompositions, systems, apparatuses and kits using the disclosed methods)and can include a digestion step. In some embodiments, the methods alsoinclude a ligating step, and the digestion step is performed prior to aligating step. In some embodiments, an amplified target sequence can bepartially digested prior to performing a ligation step. For example, anamplified target sequence can be digested by enzymatic, thermal orchemical means. In some embodiments, an amplified target sequence can bedigested prior to ligating to produce a blunt-end amplified targetsequence. In some embodiments, a blunt-ended amplified target sequencecan include a 5′ phosphate group at the 5′ end of the digested amplifiedtarget sequence.

In some embodiments, the disclosure relates generally to methods,compositions, systems, apparatuses and kits for performing multiplexnucleic acid amplification. In some embodiments, the methods (as well asrelated compositions, kits, apparatuses and systems using such methods)include amplifying one or more target sequences using one or moretarget-specific primers in the presence of polymerase underamplification conditions to produce an amplified target sequence and,ligating an adapter to the amplified target sequence. Further, themethod can include reamplifying an adapter-ligated amplified targetsequence to form a reamplified adapter-ligated amplified targetsequence. In some embodiments, a reamplified adapter-ligated amplifiedtarget sequence can be produced using no more than two rounds oftarget-specific selection.

In some embodiments, one or more target-specific primers, targetsequences or adapters can include a cleavable group. Furthermore, acleavable group can be located at a nucleotide position at, or near, theterminus of a target-specific primer, target sequence or adapter. Insome embodiments, a cleavable group can be located within 15 nucleotidesof the 3′ end or 5′ end of the nucleic acid having the cleavable group.In some embodiments, a cleavable group can be located at or near acentral nucleotide in a target-specific primer. In some embodiments, oneor more cleavable groups can be present in a target-specific primer oradapter. In some embodiments, cleavage of one or more cleavable groupsin a target-specific primer or an adapter can generate a plurality ofnucleic acid fragments with differing melting temperatures. In oneembodiment, the placement of one or more cleavable groups in atarget-specific primer or adapter can be regulated or manipulated bydetermining a comparable maximal minimum melting temperature for eachnucleic acid fragment, after cleavage of the cleavable group. In someembodiments the cleavable group can be a uracil or uridine moiety. Insome embodiments the cleavable group can be an inosine moiety. In someembodiments, at least 50% of the target-specific primers can include atleast one cleavable group. In some embodiments, each target-specificprimer includes at least one cleavable group.

In one embodiment, a multiplex nucleic acid amplification method isdisclosed herein that includes a) amplifying one or more targetsequences using one or more target-specific primers in the presence ofpolymerase to produce an amplified target sequence, and b) ligating anadapter to the amplified target sequence to form an adapter-ligatedamplified target sequence. In some embodiments, amplifying can beperformed in solution such that an amplified target sequence or atarget-specific primer is not linked to a solid support or surface. Insome embodiments, ligating can be performed in solution such that anamplified target sequence or an adapter is not linked to a solid supportor surface. In another embodiment, amplifying and ligating can beperformed in solution such that an amplified target sequence, atarget-specific primer or an adapter is not linked to a solid support orsurface.

In some embodiments, the disclosure relates generally to methods,compositions, systems, apparatuses and kits for synthesizing two or moretarget sequences within a sample. In one embodiment, the synthesizingmethod includes a) synthesizing two or more target sequences using aplurality of target-specific primers in the presence of polymerase underpolymerizing conditions to produce a plurality of synthesized targetsequences. In some embodiments, the method further includes ligating oneor more adapters to the synthesized target sequences. In someembodiments, a target sequence of interest includes one or moremutational hotspots, single nucleotide polymorphisms (SNPs), shorttandem repeats (STRs), coding regions, exons and genes. In someembodiments, the number of target sequences that can be synthesized in amultiplex reaction using the compositions (and related methods, kits,apparatuses and systems) disclosed herein can be dozens, hundreds orthousands of target sequences in a single sample. Optionally, multipletarget sequences of interest from a sample can be synthesized using oneor more target-specific primers in the presence of a polymerase underpolymerizing conditions to produce a plurality of synthesized targetsequences. In some embodiments, a synthesized target sequence can beless than 50% complementary to another synthesized target sequence. Insome embodiments, a synthesized target sequence can be substantiallynon-complementary to another target sequence in the sample. In someembodiments, a synthesized target sequence can be substantiallynoncomplementary to any one or more nucleic acid molecules in the samplethat is not a target sequence of interest. In some embodiments,synthesizing a target sequence can include ligating an adapter to asynthesized target sequence, thereby producing an adapter-ligatedsynthesized target sequence.

In some embodiments, the disclosure relates generally to synthesizing atarget sequence from a plurality of target sequences. For example, amethod of synthesizing can include synthesizing a target sequence usinga plurality of target-specific primers in the presence of polymeraseunder polymerizing conditions to produce a plurality of synthesizedtarget sequences. Synthesizing can further include resynthesizing atleast one adapter-ligated synthesized target sequence. In someembodiments, resynthesizing can include contacting at least oneadapter-ligated synthesized target sequence with at least one adapter orits complement and a polymerase under polymerizing conditions to producea plurality of resynthesized adapter-ligated synthesized targetsequences. In some embodiments, a resynthesized adapter-ligatedsynthesized target sequence can be produced using no more than tworounds of target-specific selection.

In some embodiments, a method for synthesizing target sequences caninclude a synthesizing and a ligating step. In some embodiments,ligating does not include an adapter that is substantially complementaryto a portion of a synthesized target sequence. In some embodiments, anadapter is not substantially complementary to about 30 contiguousnucleotides, or about 20 contiguous nucleotides, from a 3′ end or a 5′end of a synthesized target sequence. In some embodiments, an adaptercan include at least one sequence that is substantially complementary,or substantially identical, to at least a portion of a universal primer.

In some embodiments, the disclosure relates generally to methods,compositions, systems, apparatuses and kits for performing multiplexnucleic acid amplification. In one embodiment, the method includesamplifying one or more target sequences using one or moretarget-specific primers in the presence of polymerase underamplification conditions to produce an amplified target sequence;ligating an adapter to the amplified target sequence; and reamplifyingat least one adapter-ligated amplified target sequence. In someembodiments, reamplifying includes contacting an adapter-ligatedamplified target sequence with one or more adapters (or theircomplements) and a polymerase under amplification conditions to produceat least one reamplified adapter-ligated amplified target sequence. Insome embodiments, an amplified target sequence can be less than 50%complementary to another amplified target sequence. In some embodiments,an amplified target sequence can be substantially non-complementary toanother target sequence in the sample. In some embodiments, an amplifiedtarget sequence can be substantially noncomplementary to any one or morenucleic acid molecules in the sample that is not a target sequence ofinterest. In some embodiments, an amplified target sequence can beligated to at least one adapter, or their complement, to produce one ormore adapter-ligated amplified target sequences. In some embodiments, anadapter-ligated amplified target sequence can be reamplified to produceat least one reamplified adapter-ligated amplified target sequence. Insome embodiments, an adapter or their complement is not substantiallycomplementary to any portion of any other nucleic acid molecule withinthe sample. In some embodiments, an adapter or their complement is notsubstantially complementary to at least one amplified target sequence.In one embodiment, one or more of the adapters or their complementduring the reamplifying step can be a universal primer. In oneembodiment, the ligating step can further include ligating a DNA barcodeor DNA tagging sequence to an amplified target sequence prior toligating an adapter to an amplified target sequence.

In some embodiments, amplifying and synthesizing methods of thedisclosure can be performed as “addition-only” processes. In someembodiments, an addition-only process excludes the removal of all, or aportion of a first reaction mixture including the amplifying orsynthesizing compositions, for further manipulation during theamplification or synthesizing steps. In some embodiments, anaddition-only process can be automated for example for use inhigh-throughput processing.

In some embodiments, the disclosure generally relates to compositions(as well as related kits, methods, systems and apparatuses using thedisclosed compositions) for performing nucleic acid amplification andnucleic acid synthesis. In some embodiments, one or more of thecompositions disclosed herein (as well as related methods, kits, systemsand apparatuses) can include at least one target-specific primer and/orat least one adapter. In some embodiments, the compositions include aplurality of target-specific primers or adapters that are about 15 toabout 40 nucleotides in length. In some embodiments, the compositionsinclude one or more target-specific primers or adapters that include oneor more cleavable groups. In some embodiments, one or more types ofcleavable groups can be incorporated into a target-specific primer oradapter. In some embodiments, a cleavable group can be located at, ornear, the 3′ end of a target-specific primer or adapter. In someembodiments, a cleavable group can be located at a terminal nucleotide,a penultimate nucleotide, or any location that corresponds to less than50% of the nucleotide length of the target-specific primer or adapter.In some embodiments, a cleavable group can be incorporated at, or near,the nucleotide that is central to the target-specific primer or theadapter. For example, a target specific primer of 40 bases can include acleavage group at nucleotide positions 15-25. Accordingly, atarget-specific primer or an adapter can include a plurality ofcleavable groups within its 3′ end, its 5′ end or at a central location.In some embodiments, the 5′ end of a target-specific primer includesonly non-cleavable nucleotides. In some embodiments, the cleavable groupcan include a modified nucleobase or modified nucleotide. In someembodiments, the cleavable group can include a nucleotide or nucleobasethat is not naturally occurring in the corresponding nucleic acid. Forexample, a DNA nucleic acid can include a RNA nucleotide or nucleobase.In one example, a DNA based nucleic acid can include uracil or uridine.In another example, a DNA based nucleic acid can include inosine. Insome embodiments, the cleavable group can include a moiety that can becleaved from the target-specific primer or adapter by enzymatic,chemical or thermal means. In some embodiments, a uracil or uridinemoiety can be cleaved from a target-specific primer or adapter using auracil DNA glycosylase. In some embodiments, a inosine moiety can becleaved from a target-specific primer or adapter using hAAG or EndoV.

In some embodiments, the disclosure relates generally to compositionsincluding a target-specific primer of about 15 to about 40 nucleotidesin length having a cleavable group located near the terminus of thetarget-specific primer, hybridized to a first strand of adouble-stranded target sequence. In some embodiments, the primer issubstantially complementary to the first strand of the double-strandedtarget sequence. In some embodiments, the disclosure relates generallyto compositions including a target-specific primer of about 15 to about40 nucleotides in length having a cleavable group located near theterminus of the target-specific primer, hybridized to a first strand ofa double-stranded target sequence, and a second target-specific primerof about 15 to about 40 nucleotides in length having a cleavable grouplocated near the terminus of the second target-specific primer,hybridized to a second strand of the double-stranded target sequence. Insome embodiments, the second target-specific primer is substantiallycomplementary to the second strand of the double-stranded targetsequence.

In some embodiments, the disclosure generally relates to compositions(as well as related kits, methods, systems and apparatuses using thedisclosed compositions) for performing nucleic acid amplification andnucleic acid synthesis. In some embodiments, the compositions include atarget-specific primer of about 15 to about 40 nucleotides in lengthhaving a uracil nucleotide located near the terminus of thetarget-specific primer and a second uracil nucleotide located near thecentral nucleotide of the target-specific primer. In some embodiments,the disclosure generally relates to compositions (as well as relatedkits, methods, systems and apparatuses using the disclosed compositions)for performing nucleic acid amplification and nucleic acid synthesis. Insome embodiments, the compositions include a target-specific primer ofabout 15 to about 40 nucleotides in length having an inosine nucleotidelocated near the 3′ terminus of the target-specific primer and at leasta second inosine nucleotide located near the central nucleotide of thetarget-specific primer.

In some embodiments, the disclosure relates generally to a compositioncomprising at least one target-specific primer or at least onetarget-specific primer pair. In some embodiments, the disclosure relatesgenerally to a composition comprising a plurality of target-specificprimers. Optionally, the composition can include at least 100, 200, 300,500, 750, 1000, 1250, 1500, 1750, 2000, 2500, 3000, 4000, 5000, 7500 or10,000 target-specific primers or target-specific primer pairs. In someembodiments, the composition comprising a plurality of target-specificprimers includes at least one of the target-specific primers disclosedherein. In some embodiments, the composition comprising a plurality oftarget-specific primers includes at least one target-specific primerthat is at least 90% identical to any one of the nucleic acid sequencesprovided herein or in the concurrently filed sequencing listing. In someembodiments, the composition comprising a plurality of target-specificprimers includes one or more target-specific primer pairs disclosedherein or one or more primer pairs having at least 90% identity to anyone of the primer pair nucleic acid sequences provided herein. In someembodiments, the composition comprising a plurality of target-specificprimers can include a percentage identity of at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identity to any one or more of thenucleic acid sequences disclosed herein or in the concurrently filedsequence listing. In some embodiments, the composition comprising aplurality of target-specific primers can include any one or moretarget-specific primers selected from Tables 2, 3, 13, 14, 15, 17 and19. In some embodiments, the composition comprising a plurality oftarget-specific primers can include any one or more target-specificprimer pairs selected from Tables 2, 3, 13, 14, 15, 17 and 19. In someembodiments, the composition comprising a plurality of target-specificprimers generally relates to any one or more nucleic acid sequencesselected from SEQ ID NOs: 1-103,143 or includes at least 15 contiguousnucleotides from any one nucleic acid sequence selected from SEQ ID NOs:1-103,143. In some embodiments, the composition is generally directed toan isolated nucleic acid sequence consisting of any one or more of thenucleic acid sequences set forth in SEQ ID NOs: 1-103,143.

In some embodiments, the disclosure relates generally to a compositioncomprising a target-specific primer of about 15 nucleotides to about 40nucleotides in length. In some embodiments, the disclosure relatesgenerally to a composition comprising a plurality of at least 2target-specific primers of about 15 nucleotides to about 40 nucleotidesin length. In some embodiments, the composition comprises a plurality oftarget-specific primer pairs of about 15 nucleotides to about 40nucleotides in length designed using the primer selection criteria orprimer selection methods outlined herein.

In some embodiments, the composition includes at least onetarget-specific primer that is substantially complementary across itsentire length to at least one target sequence in a sample. In someembodiments, the composition includes a plurality of target-specificprimers, where substantially all of the plurality of target-specificprimers include a complementary nucleic acid sequence across theirentire primer lengths to one or more target sequences in a sample. Insome embodiments, the composition includes at least one target-specificprimer that is complementary across its entire length to at least onetarget sequence in a sample. In some embodiments, the compositionincludes a plurality of target-specific primers, where substantially allof the plurality of target-specific primers include a complementarynucleic acid sequence across their entire primer lengths to one or moretarget sequences in a sample.

In some embodiments, the disclosure relates generally to a compositioncomprising a plurality of target-specific primers having a cleavablegroup located at a 3′ end of at least one of the plurality of thetarget-specific primers. In some embodiments, the composition includes acleavable group located at a 3′ end of substantially all of theplurality of target-specific primers. In some embodiments, the cleavablegroup can include a uracil nucleobase, an inosine nucleoside or ananalog thereof. In some embodiments, the 3′ end of one or moretarget-specific primers can include more than one cleavable group and/ormore than one species of cleavable group. For example, a compositionhaving a cleavable group at the 3′ end of one target-specific primer caninclude one uracil moiety and an inosine moiety in the 3′ end of thesame target-specific primer. In some embodiments, the composition caninclude at least one target-specific primer that includes anon-cleavable at the 3′ terminal nucleotide. For example, atarget-specific primer can include a cleavable group at the 3′ end ofthe target-specific primer except for the terminal nucleotide at the 3′end of the target-specific primer. In some embodiments, the compositioncan include a plurality of target-specific primers where substantiallyall of the target-specific primers include a cleavable group at the 3′end except for the terminal nucleotide location.

In some embodiments, the disclosure relates generally to a compositioncomprising a plurality of target-specific primers having a cleavablegroup located near or about a central nucleotide of at least one of thetarget-specific primers. In some embodiments, the composition includes acleavable group located near or about a central nucleotide ofsubstantially all of the plurality of the target-specific primers. Forexample, in a target-specific primer of 40 nucleotides, a cleavablegroup can be located near the central nucleotide, for example at the15th nucleotide through the 25th nucleotide. In some instances, ‘near’ acentral nucleotide can refer to a percentage of the length of the entiretarget-specific primer. For example in a 40 nucleotide target-specificprimer, the location of a central cleavable group can include anylocation from about 40% to about 60% of the length of thetarget-specific primer. In some embodiments, a central nucleotide of anodd numbered target-specific primer includes the central nucleotide ofthe target-specific primer. In an even numbered target-specific primer acentral nucleotide can include one nucleotide either side of the centralnucleotide location. For example, in a 20 nucleotide target-specificprimer, the central nucleotide can include nucleotide position 10,nucleotide position 11, or both.

In some embodiments, the disclosure relates generally to a compositioncomprising a plurality of target-specific primers having at a 5′ endonly non-cleavable nucleotides. In some embodiments, the composition caninclude substantially all of the plurality of target-specific primershaving only non-cleavable nucleotides at the 5′ end. In someembodiments, the 5′ end of the plurality of target-specific primershaving only non-cleavable nucleotides can include fewer than 10nucleotides from the 5′ end. In some embodiments, the 5′ end can includefewer than 8, 7, 6, 5, 4, 3 or 2 nucleotides from the 5′ end. In someembodiments, the 5′ end having non-cleavable nucleotides can includeless than 50% of the length of the target specific primer, less than 40%of the length of the target specific primer, less than 30% of the lengthof the target specific primer, less than 20% of the length of the targetspecific primer, or less than 10% of the length of the target-specificprimer from the 5′ end.

In some embodiments, the disclosure relates generally to a compositioncomprising a plurality of target-specific primers where at least one ofthe target-specific primers includes less than 20% of the nucleotidesacross the primer's entire length containing a cleavable group. In someembodiments, the composition comprises a plurality of target-specificprimers where substantially all of the target-specific primers includeless than 20% of the nucleotides across each primer's entire lengthcontaining a cleavable group. For example, a target-specific primer of20 nucleotides in length can include 4 or fewer cleavage groups. In someembodiments, the disclosure relates generally to a compositioncomprising a plurality of target-specific primers where at least one ofthe target-specific primers includes less than 10% of the nucleotidesacross the primer's entire length containing a cleavable group. In someembodiments, the composition comprises a plurality of target-specificprimers where substantially all of the target-specific primers includeless than 10% of the nucleotides across each primer's entire lengthcontaining a cleavable group. For example, a target-specific primer of20 nucleotides in length can include 2 or fewer cleavage groups.

In some embodiments, the disclosure relates generally to a compositioncomprising a plurality of target-specific primers having minimalcross-hybridization to at least one of the target-specific primers inthe plurality of primers. In some embodiments, the disclosure relatesgenerally to a composition comprising a plurality of target-specificprimers having minimal cross-hybridization to substantially all of thetarget-specific primers in the plurality of primers. In someembodiments, minimal cross-hybridization to one or more target-specificprimers in the plurality of primers can be evaluated by the formation ofprimer-dimers or dimer-dimers. In some embodiments, the composition caninclude fewer primer-dimers in a multiplex PCR amplification reaction ascompared to a multiplex PCR amplification reaction of the prior artunder corresponding amplification conditions.

In some embodiments, the disclosure relates generally to a compositioncomprising a plurality of target-specific primers, where at least one ofthe target-specific primers includes minimal cross-hybridization tonon-specific sequences present in a sample. In some embodiments, thecomposition comprises a plurality of target-specific primers wheresubstantially all of the target-specific primers include minimalcross-hybridization to non-specific sequences present in a sample. Insome embodiments, minimal cross-hybridization to non-specific sequencespresent in a sample can be evaluated by the presence of ‘percent ofreads off-target’ or a decrease in ‘percent of reads on target’. In someembodiments, the compositions as disclosed herein can provide fewer‘percent of reads off-target’ or an increase in ‘percent of reads ontarget’ in multiplex PCR amplification reactions as compared tomultiplex PCR amplification reactions of the prior art undercorresponding amplification conditions. The “plex” of a given multiplexamplification refers generally to the number of differenttarget-specific sequences that are amplified during a single multiplexamplification according to the disclosure. In some embodiments, the plexcan be about 12-plex, 24-plex, 48-plex, 96-plex, 192-plex, 384-plex,768-plex, 1536-plex, 3072-plex, 6144-plex or higher. In someembodiments, minimal cross-hybridization to non-specific sequencespresent in a sample can include less than 15%, less than 12%, or fewerthan 10% reads off target. In some embodiments, the percent of reads ontarget per multiplex amplification can be greater than 85%, 88%, 90%,92%, 94%, 95%, 96%, 97%, 98%, or more.

In some embodiments, the disclosure relates generally to a compositioncomprising a plurality of target-specific primers having minimalself-complementarity. In some embodiments, the composition includes atleast one target-specific primer that does not form a secondarystructure, such as loops or hairpins. In some embodiments, thecomposition includes a plurality of target-specific primers where amajority (i.e., greater than 50%), or substantially all of the pluralityof target-specific primers fail to form a secondary structure. The“plex” of a given multiplex amplification refers generally to the numberof different target-specific sequences that are amplified during asingle multiplex amplification according to the disclosure. In someembodiments, the plex can be about 12-plex, 24-plex, 48-plex, 96-plex,192-plex, 384-plex, 768-plex, 1536-plex, 3072-plex, 6144-plex or higher.In some embodiments, minimal self-complementarity can include less than10%, less than 8%, less than 5% or less than 3% of the plurality oftarget-specific primers possessing self-complementarity that allows atarget-specific primer to form a secondary structure.

In some embodiments, the disclosure relates generally to a compositioncomprising a plurality of target-specific primers having minimalnucleotide sequence overlap at a 3′ end or a 5′ end. In someembodiments, the composition can include minimal overlap of nucleotidesequence in the 3′ end of at least one target-specific primer. In someembodiments, the composition can include minimal overlap of nucleotidesequence in the 3′ end of substantially all of the plurality oftarget-specific primers. In some embodiments, the composition caninclude minimal overlap of nucleotide sequence in the 5′ end of at leastone target-specific primer. In some embodiments, the composition caninclude minimal overlap of nucleotide sequence in the 5′ end ofsubstantially all of the plurality of target-specific primers. In someembodiments, the composition can include minimal overlap of nucleotidesequence in the 3′ end and the 5′ end of at least one target-specificprimer. In some embodiments, the composition can include minimal overlapof nucleotide sequence in the 3′ end and the 5′ end of substantially allof the plurality of target-specific primers. In some embodiments, theamount of nucleotide sequence overlap between one or moretarget-specific primers is less than 8 nucleotides. In some embodiments,the amount of nucleotide sequence overlap between one or moretarget-specific primers is less than 5 nucleotides. In some embodiments,the amount of nucleotide sequence between one or more target-specificprimers of the plurality of primers is less than 8, 7, 6, 5, 4, 3, 2 or1 nucleotide. In some embodiments, the composition can include aplurality of target-specific primers including a nucleotide sequence gapof one or more nucleotides. In some embodiments, the composition caninclude a nucleotide sequence gap of 1, 2, 3, 4, 5, 10, 15, 20 or morenucleotides between two or more of the plurality of target-specificprimers. In some embodiments, the composition can include a nucleotidesequence gap of about 50 nucleotides between two or more target-specificprimers in the plurality of target-specific primers. In someembodiments, the composition can include a nucleotide sequence gap ofabout 10, 20, 30, 40, or 50 nucleotides between substantially all of thetarget-specific primers in the plurality of target-specific primers.

In some embodiments, the disclosure relates generally to a compositioncomprising a plurality of target-specific primers of about 15nucleotides to about 40 nucleotides in length having at least two ormore following criteria: a cleavable group located at a 3′ end ofsubstantially all of the plurality of primers, a cleavable group locatednear or about a central nucleotide of substantially all of the pluralityof primers, substantially all of the plurality of primers at a 5′ endincluding only non-cleavable nucleotides, minimal cross-hybridization tosubstantially all of the primers in the plurality of primers, minimalcross-hybridization to non-specific sequences present in a sample,minimal self-complementarity, and minimal nucleotide sequence overlap ata 3′ end or a 5′ end of substantially all of the primers in theplurality of primers. In some embodiments, the composition can includeany 3, 4, 5, 6 or 7 of the above criteria.

In some embodiments, the disclosure relates generally to a compositioncomprising a plurality of at least 2 target-specific primers of about 15nucleotides to about 40 nucleotides in length having two or more of thefollowing criteria, a cleavable group located near or about a centralnucleotide of substantially all of the plurality of primers,substantially all of the plurality of primers at a 5′ end including onlynon-cleavable nucleotides, substantially all of the plurality of primershaving less than 20% of the nucleotides across the primer's entirelength containing a cleavable group, at least one primer having acomplementary nucleic acid sequence across its entire length to a targetsequence present in a sample, minimal cross-hybridization tosubstantially all of the primers in the plurality of primers, minimalcross-hybridization to non-specific sequences present in a sample, andminimal nucleotide sequence overlap at a 3′ end or a 5′ end ofsubstantially all of the primers in the plurality of primers. In someembodiments, the composition can include any 3, 4, 5, 6 or 7 of theabove criteria.

In some embodiments, the disclosure relates generally to a compositioncomprising a plurality of target-specific primers designed according tothe criteria disclosed here or including any one or more of thetarget-specific primers disclosed herein, where at least one of theplurality of target-specific primers is substantially complementaryacross its entire length to at last a portion of one or more genesselected from ABI1; ABL1; ABL2; ACSL3; ACSL6; AFF1; AFF3; AFF4; AKAP9;AKT1; AKT2; ALK; APC; ARHGAP26; ARHGEF12; ARID1A; ARNT; ASPSCR1; ASXL1;ATF1; ATIC; ATM; AXIN2; BAP1; BARD1; BCAR3; BCL10; BCL11A; BCL11B; BCL2;BCL3; BCL6; BCL7A; BCL9; BCR; BIRC3; BLM; BMPR1A; BRAF; BRCA1; BRCA2;BRD3; BRD4; BRIP1; BUB1B; CARD11; CARS; CASC5; CBFA2T3; CBFB; CBL; CBLB;CBLC; CCDC6; CCNB1IP1; CCND1; CCND2; CD74; CD79A; CDC73; CDH1; CDH11;CDK4; CDK6; CDKN2A; CDKN2B; CDKN2C; CDX2; CEBPA; CEP110; CHEK1; CHEK2;CHIC2; CHN1; CIC; CIITA; CLP1; CLTC; CLTCL1; COL1A1; CREB1; CREB3L2;CREBBP; CRTC1; CRTC3; CSF1R; CTNNB1; CXCR7; CYLD; CYTSB; DCLK3; DDB2;DD1T3; DDR2; DDX10; DDX5; DDX6; DEK; DGKG; DICER1; DNMT3A; EGFR; EIF4A2;ELF4; ELL; ELN; EML4; EP300; EPS15; ERBB2; ERBB4; ERC1; ERCC2; ERCC3;ERCC4; ERCC5; ERG; ETV1; ETV4; ETV5; ETV6; EWSR1; EXT1; EXT2; EZH2;FAM123B; FANCA; FANCC; FANCD2; FANCE; FANCF; FANCG; FAS; FBXW7; FCRL4;FGFR1; FGFR10P; FGFR2; FGFR3; FH; FIP1L1; FLCN; FLI1; FLT1; FLT3; FNBP1;FOXL2; FOXO1; FOXO3; FOXO4; FOXP1; FUS; GAS7; GATA1; GATA2; GATA3; GMPS;GNAQ; GNAS; GOLGA5; GOPC; GPC3; GPHNGPR124; HIP1; HIST1H4I; HLF; HNF1A;HNRNPA2B1; HOOK3; HOXA11; HOXA13; HOXA9; HOXC11; HOXC13; HOXD13; HRAS;HSP90AA1; HSP90AB1; IDH1; IDH2; IKZF1; IL2; IL21R; IL6ST; IRF4; ITGA10;ITGA9; ITK; JAK1; JAK2; JAK3; KDM5A; KDM5C; KDM6A; KDR; KDSR; KIAA1549;KIT; KLF6; KLK2; KRAS; KTN1; LASP1; LCK; LCP1; LHFP; LIFR; LMO2; LPP;MAF; MALT1; MAML2; MAP2K1; MAP2K4; MDM2; MDM4; MECOM; MEN1; MET; MITF;MKL1; MLH1; MLL; MLLT1; MLLT10; MLLT3; MLLT4; MLLT6; MN1; MPL; MRE11A;MSH2; MSH6; MSI2; MSN; MTCP1; MTOR; MUC1; MYB; MYC; MYCL1; MYCN; MYH11;MYH9; MYST3; MYST4; NACA; NBN; NCOA1; NCOA2; NCOA4; NEK9; NF1; NF2;NFE2L2; NFKB2; NIN; NKX2-1; NLRP1; NONO; NOTCH1; NOTCH2; NPM1; NR4A3;NRAS; NSD1; NTRK1; NTRK3; NUMA1; NUP214; NUP98; OLIG2; OMD; PAFAH1B2;PALB2; PATZ1; PAX3; PAX5; PAX7; PAX8; PBRM1; PBX1; PCM1; PDE4DIP; PDGFB;PDGFRA; PDGFRB; PER1; PHOX2B; PICALM; PIK3CA; PIK3R1; PIM1; PLAG1; PML;PMS1; PMS2; POU2AF1; POU5F1; PPARG; PPP2R1A; PRCC; PRDM16; PRF1;PRKAR1A; PRRX1; PSIP1; PTCH1; PTEN; PTPN11; RABEP1; RAD50; RAD51L1;RAF1; RANBP17; RAP1GDS1; RARA; RB1; RBM15; RECQL4; REL; RET; RHOH;RNF213; ROS1; RPN1; RPS6KA2; RUNX1; RUNX1T1; SBDS; SDHAF2; SDHB; SETD2;SFPQ; SFRS3; SH3GL1; SLC45A3; SMAD4; SMARCA4; SMARCB1; SMO; SOCS1; SRC;SRGAP3; SS18; SS18L1; STIL; STK11; STK36; SUFU; SYK; TAF15; TAF1L; TAL1;TAL2; TCF12; TCF3; TCL1A; TET1; TET2; TEX14; TFE3; TFEB; TFG; TFRC;THRAP3; TLX1; TLX3; TMPRSS2; TNFAIP3; TOP1; TP53; TPM3; TPM4; TPR;TRIM27; TRIM33; TRIP11; TSC1; TSC2; TSHR; USP6; VHL; WAS; WHSC1L1; WRN;WT1; XPA; XPC; ZBTB16; ZMYM2; ZNF331; ZNF384; and ZNF521.

In some embodiments, the disclosure relates generally to a compositioncomprising a plurality of target-specific primers designed according tothe criteria disclosed here or including any one or more of thetarget-specific primers disclosed herein, where at least one of theplurality of target-specific primers is substantially complementaryacross its entire length to at last a portion of one or more genesselected from ABL1; AKT1; ALK; APC; ATM; BRAF; CDH1; CDKN2A; CSF1R;CTNNB1; EGFR; ERBB2; ERBB4; FBXW7; FGFR1; FGFR2; FGFR3; FLT3; GNAS;HNF1A; HRAS; IDH1; JAK2; JAK3; KDR; KIT; KRAS; MET; MLH1; MPL; NOTCH1;NPM1; NRAS; PDGFRA; PIK3CA; PTEN; PTPN11; RB1; RET; SMAD4; SMARCB1; SMO;SRC; STK11; TP53; and VHL.

In some embodiments, the disclosure relates generally to a compositioncomprising a plurality of target-specific primers designed according tothe criteria disclosed here or including any one or more of thetarget-specific primers disclosed herein, where at least one of theplurality of target-specific primers is substantially complementaryacross its entire length to at last a portion of one or more genesselected from ABCA4; ABCC8; ABCD1; ACADVL; ACTA2; ACTC; ACTC1; ACVRL1;ADA; AIPL1; AIRE; ALK1; ALPL; AMT; APC; APP; APTX; AR; ARL6; ARSA; ASL;ASPA; ASS; ASS1; ATL; ATM; ATP2A2; ATP7A; ATP7B; ATXN1; ATXN2; ATXN3;ATXN7; BBS6; BCKDHA; BCKDHB; BEST1; BMPR1A; BRCA1; BRCA2; BRIP1; BTD;BTK; C2orf25; CA4; CALR3; CAPN3; CAV3; CCDC39; CCDC40; CDH23; CEP290;CERKL; CFTR; CHAT; CHD7; CHEK2; CHM; CHRNA1; CHRNB1; CHRND; CHRNE;CLCN1; CNBP; CNGB1; COH1; COL11A1; COL11A2; COL1A1; COL1A2; COL2A1;COL3A1; COL4A5; COL5A1; COL5A2; COL7A1; COL9A1; CRB1; CRX; CTDP1; CTNS;CYP21A2; CYP27A1; DAX1; DBT; DCX; DES; DHCR7; DJ1; DKC1; DLD; DMD; DMPK;DNAAF1; DNAAF2; DNAH11; DNAH5; DNA11; DNAI2; DNAL1; DNM2; DOK7; DSC2;DSG2; DSP; DYSF; DYT1; EMD; ENG; EYA1; EYS; F8; F9; FANCA; FANCC; FANCF;FANCG; FANCJ; FANDC2; FBN1; FBXO7; FGFR1; FGFR3; FMO3; FMR1; FOXL2;FRG1; FRMD7; FSCN2; FXN; GAA; GALT; GBA; GBE1; GCSH; GDF5; GJB2; GJB3;GJB6; GLA; GLDC; GNE; GNPTAB; GPC3; GPR143; GUCY2D; HBA1; HBA2; HBB; HD;HERG; HEXA; HFE; HHF; HIBCH; HLA-B27; HMBS; HPLH1; HPRP3; HR; HTNB; HTT;IKBKAP; IKBKG; IL2RG; IMPDH1; ITGB4; JAG1; JPH3; KCNE1; KCNE2; KCNH2;KCNQ1; KCNQ4; KIAA0196; KLHL7; KRAS; KRT14; KRT5; L1CAM; LAMB3; LAMP2;LDB3; LMNA; LMX18; LRAT; LRRK2; MAPT; MC1R; MECP2; MED12; MEN1; MERTK;MFN2; MKKS; MLH1; MMAA; MMAB; MMACHC; MMADHC; MPZ; MSH2; MTM1; MTND5;MTTG; MTTI; MTTK; MTTL1; MTTQ; MUT; MYBPC3; MYH11; MYH6; MYH7; MYL2;MYL3; MYLK2; MYO7A; ND5; ND6; NEMO; NF1; NF2; NIPBL; NR0B1; NR2E3; NRAS;NSD1; OCA2; OCRL; OPA1; OTC; PABPN1; PAFAH1B1; PAH; PARK2; PARK7;PARKIN; PAX3; PAX6; PCDH15; PEX1; PEX2; PEX10; PEX13; PEX14; PEX19;PEX26; PEX3; PEX5; PINK1; PKD1; PKD2; PKD3; PKHD1; PKP2; PLEC1; PLOD1;PMM2; PMP22; POLG; PPT1; PRCD; PRKAG2; PRNP; PROM1; PRPF3; PRPF8; PRPH2;PRPN; PSEN1; PSEN2; PTCH1; PTPN11; RAB7A; RAF1; RA11; RAPSN; RBI; RDH12;RDS; RECQL3; RET; RHO; ROR2; RP1; RP2; RP9; RPE65; RPGR; RPGRIP1; RPL11;RPL35A; RPS10; RPS17; RPS19; RPS24; RPS26; RPS6KA3; RPS7; RPSL5; RS1;RSPH4A; RSPH9; RYR1; RYR2; SALL4; SCA3; SCN5A; SCN9A; SEMA4A; SERPINA1;SERPING1; SGCD; SH3BP2; SHOX; SIX1; SIX5; SLC25A13; SLC25A4; SLC26A4;SMAD4; SMN1; SNCA; SNRNP200; SOD1; SOS1; SOX9; SP110; SPAST; SPATA7;SPG3A; SPG4; SPG7; TAF1; TBX5; TCOF1; TGFBR1; TGFBR2; TNFRSC13C; TNNC1;TNNI3; TNNT1; TNNT2; TNXB; TOPORS; TOR1A; TP53; TPM1; TRNG; TRNI; TRNK;TRNL1; TRNQ; TSC1; TSC2; TTN; TTPA; TTR; TULP1; TWIST1; TXNDC3; TYR;USH1C; USH1H; USH2A; VCL; VHL; VPS13B; WAS; WRN; WT1; and ZNF9.

In some embodiments, the disclosure relates generally to a compositioncomprising a plurality of target-specific primers designed according tothe criteria disclosed here or including any one or more of thetarget-specific primers disclosed herein, where at least one of theplurality of target-specific primers is substantially complementaryacross its entire length to at last a portion of one or more genesassociated with breast cancer selected from AIM1, AR, ATM, BARD1, BCAS1,BRIP1, CCND1, CCND2, CCNE1, CDH1, CDK3, CDK4, CDKN2A, CDKN2B, CAMK1D,CHEK2, DIRAS3, EGFR, ERBB2, EPHA3, ERBB4, ETV6, GNRH1, KCTD9, CDCA2,EBF2, EMSY, BNIP3L, PNMA2, DPYSL2, ADRA1A, STMN4, TRIM35, PAK1, AQP11,CLSN1A, RSF1, KCTD14, THRSP, NDUFC2, ALG8, KCTD21, USP35, GAB2, DNAH9,ZNF18, MYOCD, STK11, TP53, JAK1, JAK2, MET, PDGFRA, PML, PTEN, RET,TMPRSS2, WNK1, FGFR1, IGF1R, PPP1R12B, PTPRT, GSTM1, IPO8, MYC, ZNF703,MDM1, MDM2, MDM4, MKK4, P14 KB, NCOR1, NBN, PALB2, RAD50, RAD51, PAK1,RSF1, INTS4, ZMIZ1, SEPHS1, FOXM1, SDCCAG1, IGF1R, TSHZ2, RPSK6K1,PPP2R2A, MTAP, MAP2K4, AURKB, BCL2, BUB1, CDCA3, CDCA4, CDC20, CDC45,CHEK1, FOXM1, HDAC2, IGF1R, KIF2C, KIFC1, KRAS, RBI, SMAD4, NCOR1, UTX,MTHDFD1L, RAD51AP1, TTK and UBE2C.

In some embodiments, the disclosure is generally related to acombination of polynucleotides, where the combination of polynucleotidesincludes at least one polynucleotide selected from Tables 2, 3, 13, 14,15, 17 and 19, and one or more additional polynucleotides independent ofthe polynucleotides disclosed herein. In some embodiments, thedisclosure is generally related to a combination of polynucleotides,where the combination of polynucleotides includes a polynucleotidehaving at least 90% identity to one or more polynucleotides selectedfrom Tables 2, 3, 13, 14, 15, 17 and 19. In some embodiments, thedisclosure relates to 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 200, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000,8000, 9000, 10,000 or more polynucleotides selected from Tables 2, 3,13, 14, 15, 17 and 19.

In some embodiments, the disclosure is generally related to acombination of polynucleotides, where the combination of polynucleotidesincludes at least one polynucleotide selected from Tables 2, 3, 13, 14,15, 17 and 19, and one or more additional polynucleotides independent ofthe polynucleotides disclosed herein. In some embodiments, thedisclosure is generally related to a combination of polynucleotides,where the combination of polynucleotides includes at least onepolynucleotide having at least 90% identity to one or morepolynucleotides selected from Tables 2, 3, 13, 14, 15, 17 and 19. Insome embodiments, the disclosure relates to 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000, 2000, 3000, 4000,5000, 6000, 7000, 8000, 9000, 10,000 or more polynucleotides selectedfrom Tables 2, 3, 13, 14, 15, 17 and 19 or one or more polynucleotideshaving at least 90% identity thereto.

In some embodiments, the disclosure is generally related to a pair ofpolynucleotides that specifically anneal to a portion of at least onegene selected from EGFR, BRAF or KRAS. In one embodiment, a pair ofpolynucleotides that specifically anneal to a portion of the EGFR geneincludes any one or more of the following Amplicon IDs: 229910389,227801665, 229055506, 230397881, 230175199, 230195609, 228630698,230632980, 227722022, 232978808, 231616816, 230481741, 231198336,229919273, 227816834, 228030652, 230679876, 229747025, 228741519,228636601, 230635054, 230738160, 232984355, 228941652, 230495367,231212482, 229608278, 230461276, 228035285, 230683371, 230173849,330137554, 228857751, 230742871, 232237229, 228956984, 228732632,231222418, 231493149, 229630617, 229052979, 230392156, 230683680,230187475, 228709018, 230628101, 227716821, 227830783, 232260099,230075336, 231314233, and 231239581. In one embodiment, a pair ofpolynucleotides that specifically anneal to a portion of the BRAF geneincludes any one or more of the following Amplicon IDs: 222636793,223460541, 223967627, 326913823, 223739184, 223944056, 224404546,222922922, 224119138, 223519358, 223465859, 223971374, 222680486,223741661, 223950351, 224410546, 222935598, 224119999, 222629880,223175118, 223719489, 225222024, 222684242, 223700378, 222258987,222895407, 223103332, 222635553, 223177865, 223960162, 326889377,223588249, 223708886, 222259284, 222903910, and 223104608. In oneembodiment, a pair of polynucleotides that specifically anneal to aportion of the KRAS gene includes any one or more of the followingAmplicon IDs: 233361228, 234355242, 234355242, 233466735, 233466735,231132733, 231132733, 234764991, 234764991, 233467720, 233467720,231133990, 231133990, 233356818, 326772204, and 326772204.

In some embodiments, the disclosure is generally related to kits (aswell as related compositions, methods, apparatuses and systems usingsuch kits) for amplifying one or more target sequences in a sample. Insome embodiments, the kits for amplifying one or more target sequencesin a sample include at least one target-specific primer that can amplifythe at least one target sequence in the sample. In some embodiments, thekit can include at least two target-specific primers that can amplify atleast one target sequence in the sample. In another embodiment, the kitcan include a plurality of target-specific primers for amplifying atleast two target sequences in a sample, where the kit includes a) afirst target-specific primer having at least 90% identity to a nucleicacid sequence selected from SEQ ID NOs: 1-103,143 being substantiallycomplementary to the first target sequence in a sense direction; b) asecond target-specific primer having at least 90% identity to a nucleicacid sequence selected from SEQ ID NOs: 1-103,143 being substantiallycomplementary to the first target sequence in a antisense direction; c)a third target-specific primer having at least 90% identity to a nucleicacid sequence selected from SEQ ID NOs: 1-103,143 being substantiallycomplementary to the second target sequence in a sense direction; and,d) a fourth target-specific primer having at least 90% identity to anucleic acid sequence selected from SEQ ID NOs: 1-103,143 beingsubstantially complementary to the second target sequence in a antisensedirection. In some embodiments, the sample can be an environmental,aquatic, microbiological, entomological, plant, fungi, animal ormammalian nucleic acid containing sample. In some embodiments, thesample can include a clinical, surgical, physician, forensic orlaboratory obtained nucleic acid sample.

In some embodiments, the disclosure relates generally to a method foramplifying a plurality of target sequences in a sample comprisingcontacting at least some portion of the sample with at least onetarget-specific primer as disclosed herein, or designed using the primerselection criteria disclosed herein, and a polymerase underamplification conditions thereby producing at least one amplified targetsequence. In some embodiments, the method further includes ligating atleast one adapter to at least one amplified target sequence, therebyproducing at least one adapter-ligated amplified target sequence. Insome embodiments, the method includes any one or more of thetarget-specific primers provided in Tables 2, 3, 13, 14, 15, 17 and 19or any nucleic acid sequence having at least 90% identity to any one ormore of the target-specific primers provided in Tables 2, 3, 13, 14, 15,17 and 19.

In some embodiments, the disclosure is generally related to anamplification product generated by amplifying at least one targetsequence present in a sample with one or more target-specific primersdisclosed herein or one or more target-specific primers designed usingthe primer selection criteria disclosed herein. In some embodiments, thedisclosure is generally related to an amplification product generated bycontacting at least one target sequence in a sample with one or moretarget-specific primers disclosed herein or one or more target-specificprimers designed using the primer selection criteria disclosed hereinunder amplification conditions. In some embodiments, the amplificationproduct can include one or more mutations associated with cancer orinherited disease. For example, a sample suspected of containing one ormore mutations associated with at least one cancer can be subjected toany one of the amplification methods disclosed herein. The amplificationproducts obtained from the selected amplification method can optionallybe compared to a normal or matched sample known to be noncancerous withrespect to the at least one cancer, and can therefore be used as areference sample. In some embodiments, the amplification productsobtained by the methods disclosed herein can be optionally sequencedusing any suitable nucleic acid sequencing platform to determine thenucleic acid sequence of the amplification products, and optionallycompared to sequencing information from the normal or non-canceroussample. In some embodiments, amplification products can include one ormore markers associated with antibiotic resistance, pathogenicity orgenetic modification. In some embodiments, nucleic acid sequences of oneor more amplification products obtained by contacting at least onetarget sequence with at least one target-specific primer underamplification conditions can be used to determine the presence orabsence of a genetic variant within the one or more amplificationproducts.

In some embodiments, the disclosure generally relates to compositions(as well as related kits, methods, systems and apparatuses using thedisclosed compositions) for performing nucleic acid amplification andnucleic acid synthesis. In some embodiments, the composition includes aplurality of target-specific primer pairs, at least one target-specificprimer pair including a target-specific forward primer and atarget-specific reverse primer. In some embodiments, the compositionincludes at least 100, 200, 500, 750, 1000, 2500, 5000, 7500, 10000,12000, 15000, 17500, 20000 or 50000 different primer pairs, some or allof which can be target-specific. Optionally at least two of thedifferent target-specific primer pairs are directed to (i.e., arespecific for) different target sequences.

In some embodiments, the composition includes at least onetarget-specific primer pair that can be specific for at least oneamplified target sequence. In some embodiments, the composition includesa plurality of target-specific primer pairs, at least twotarget-specific primer pairs being specific for at different amplifiedtarget sequences. In some embodiments, the composition includes atarget-specific primer pair, which each member of the primer pairincludes a target-specific primer that can hybridize to at least aportion of a first amplified sequence or its complement, and that issubstantially non-complementary to the 3′ end or the 5′ end of any otheramplified sequence in the sample. In some embodiments, the compositionincludes at least one target-specific primer pair that can besubstantially non-complementary to a portion of any other nucleic acidmolecule in the sample. In some embodiments, the compositions include aplurality of target-specific primer pairs that include one or morecleavable groups at one or more locations within the target-specificprimer pair.

In some embodiments, the composition includes one or moretarget-specific primer pairs that can amplify a short tandem repeat,single nucleotide polymorphism, gene, exon, coding region, exome, orportion thereof. For example, a plurality of target-specific primerpairs can uniformly amplify one gene, exon, coding region, exome orportion thereof. In some embodiments, the compositions includetarget-specific primer pairs designed to minimize overlap of nucleotidesequences amplified using the one or more target-specific primer pairs.In some embodiments, the nucleotide sequence overlap between one or moretarget-specific primers can be minimized at the 3′ end, the 5′ end, orboth. In some embodiments, at least one primer in a plurality oftarget-specific primers includes less than 5 nucleotides of nucleotidesequence overlap at the 3′ end, 5′ end or both. In some embodiments, atleast one target-specific primer of a plurality of target-specificprimers includes a nucleotide sequence gap of at least one nucleotide,as compared to the plurality of target-specific primers. In someembodiments, the compositions include one or more target-specific primerpairs designed to comprehensively amplify one or more genes or exons.For example, a plurality of target-specific primer pairs can be designedto uniformly amplify (i.e., provide 100% representation of allnucleotides) in a single gene or exon.

In some embodiments, at least two pairs of target-specific primers arecapable of hybridizing to locations on a template nucleic acid andserving as substrates for template-dependent primer extension by apolymerase. In some embodiments, the template-dependent primer extensioncan include amplification of the region of template located between thesites of hybridization of the primers of the at least two pairs ofprimers, resulting in formation of an amplified region or “amplicon”.Typically, the sequence of the amplicon includes the sequence of thetemplate located between the sites of hybridization of the primers, aswell as at least part of the sequence of the primers themselves. In someembodiments, the amplification reaction can include at least about 5,10, 25, 50, 100, 150, 200, 250, 400, 500, 750, 1000, 1200, 1250, 1500,1750, 2000, 2250, 2500, 2750, 3000, 5000, 7500 or 10,000 differentprimer pairs. In some embodiments, the amplification reaction can resultin the generation of at least about 5, 10, 25, 50, 100, 150, 200, 250,400, 500, 750, 1000, 1200, 1250, 1500, 1750, 2000, 2250, 2500, 2750,3000, 5000, 7500 or 10,000 different amplicons. In some embodiments, atleast about 75%, 80%, 90%, 95%, 97% or 99% of the amplicons generatedduring the amplification reaction are similarly sized, for example, theamplicons differ in size from each other by no more than 5, 10, 25, 50,75, 100, 500, 1000 or 2000 nucleotides. In some embodiments, thedifference in length between any two amplicons is no greater than 1%,5%, or 10% of average amplicon length in the amplification reactionmixture. Optionally, the average amplicon length is about 50, 60, 70,80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 500, 1000, 2000, 10,000nucleotides or greater. In some embodiments, the standard deviation inlength among amplicons in a mixture is no greater than 0.1, 0.25, 0.4,0.5, 0.75, 1, 1.5, 2.0, 2.4 or 3.0

In some embodiments, the compositions include target-specific primerpairs designed to generate amplified target sequences that overlap withan adjacent amplified target sequence by a single nucleotide. In someembodiments, the compositions include target-specific primer pairsdesigned to generate an amplified target sequence that does not overlapwith an adjacent amplified target sequence. For example, target-specificprimer pairs can be designed to generate amplified target sequences thatare separated by one or more nucleotides. In some embodiments, thecomposition includes target-specific primer pairs designed to separateamplified target sequences by about 50 nucleotides.

In some embodiments, the composition includes a plurality of exon- orgene-specific, target-specific primer pairs that can be substantiallycomplementary to an individual exon or gene. In some embodiments, thecomposition includes a plurality of exon- or gene-specific,target-specific primer pairs that can be substantially complementary toone or more exons or genes. In some embodiments, the compositionincludes a plurality of substantially complementary exon- orgene-specific, target-specific primer pairs and that no two primer pairsamplify greater than 10% of the same target sequence. In someembodiments, no two target-specific primer pairs amplify the same exonor gene. In some embodiments, the target-specific primer pairs amplifyabout 100 to about 600 nucleotides of a target sequence. In someembodiments, the target-specific primer pairs can be used to amplifyabout 25% to 100% of an exon, gene or coding region. In someembodiments, the compositions includes a plurality of target-specificprimer pairs to generate a plurality of amplified target sequences andthat no individual amplified target sequence is overexpressed by morethan 50% as compared to the other amplified target sequences. In someembodiments, the compositions includes a plurality of target-specificprimer pairs designed to generate a plurality of amplified targetsequences that are substantially homogenous (i.e., homogenous withrespect to GC content, melting temperature, or amplified target sequencelength). In some embodiments, the plurality of target-specific primerpairs overlap in sequence by no more than five nucleotides.

In some embodiments, the disclosure generally relates to a method forpreventing or eliminating non-specific amplification products in amultiplex PCR reaction. In some embodiments, the method includes (aswell as related compositions, kits, systems and apparatuses used usingthe disclosed methods) hybridizing one or more target-specific primerpairs to a target sequence in a sample having a plurality of targetsequences, extending the hybridized target-specific primers to form aplurality of amplified target sequences, denaturing and annealing theamplified target sequences to form a plurality of double-strandedamplified target sequences and performing a digesting step on the samplecontaining the double-stranded amplified target sequences to eliminatenon-specific amplification products. In some embodiments, the methodincludes one or more cleavable groups at one or more locations in one ormore target-specific primer pairs. In some embodiments, eachtarget-specific primer pair includes at least one cleavable group. Insome embodiments, each target-specific primer of the primer pairsincludes a cleavable group. In some embodiments, the digestion is anenzymatic or chemical digestion. In some embodiments, the digestion stepincludes partial digestion of a target-specific primer of an amplifiedtarget sequence. In some embodiments, the method includes a thermostablepolymerase. In some embodiments, the thermostable polymerase can beoptionally reactivated by heat or chemical treatment.

In some embodiments, the composition includes a plurality oftarget-specific primer pairs directed to one or more diseases ordisorders. In some embodiments, a target-specific primer pair can besubstantially complementary to a target sequence correlated orassociated with one or more cancers. In some embodiments, atarget-specific primer pair can be substantially complementary to atarget sequence correlated with or associated with one or morecongenital or inherited disorders. In some embodiments, one or moretarget-specific primer pairs can be associated with one or moreneurological, metabolic, neuromuscular, developmental, cardiovascular orautoimmune disorders. In some embodiments, one or more target-specificprimer pairs can be associated with one or more genes or exonsassociated with one or more neurological, metabolic, neuromuscular,developmental, cardiovascular or autoimmune disorders. In someembodiments, the plurality of target-specific primers can include a geneor gene fragment associated with neoplastic development in mammals.

In some embodiments, the disclosure relates generally to compositions(as well as related kits, methods, systems and apparatuses using thedisclosed compositions) comprising any, some or all of the primersdisclosed herein, including in the Examples and in the relatedappendices, supplements and tables attached hereto. In some embodiments,the disclosure relates generally to compositions (as well as relatedkits, methods, systems and apparatuses using the disclosed compositions)comprising any of the primer pools used in the Examples, or any subsetthereof. For example, in some embodiments the disclosure relatesgenerally to compositions including one or more target-specific primersselected from the primers listed in Tables 2, 3, 13, 14, 15, 17 and 19,which include sets of primers designed and selected using the designmethods and selection criteria of the disclosure, and which have beenused to perform highly multiplex amplification according to the methodsdisclosed herein. It will be readily appreciated by one of ordinaryskill in the art that any subsets of each of the primer sets set forthin Tables 2, 3, 13, 14, 15, 17 and 19 can also be expected to supportmultiplex amplification, since the entire set of primers of each tablehas been demonstrated to support such multiplex amplification andremoval of particular primer pairs from the pool will not be expected tosignificantly alter the performance of the remaining primers forpurposes of multiplex amplification. In some embodiments, the disclosurerelates generally to compositions including any 1, 2, 5, 10, 25, 50,100, 250, 500, 750, 1000, 2500, 5000, 7500, 10000, 12500, 50000, 100000or more different target-specific primer pairs set forth in Tables 2, 3,13, 14, 15, 17 and 19. In some embodiments, the disclosure relates to acomposition including at least 1, 2, 5, 10, 25, 50, 100, 250, 500, 750,1000, 2500, 5000, 7500, 10000, 12500, 50000, 100000 or more primersselected from Tables 2, 3, 13, 14, 15, 17 and 19, or their complements.In some embodiments, the disclosure relates to a composition includingat least 1, 2, 5, 10, 25, 50, 100, 250, 500, 750, 1000, 2500, 5000,7500, 10000, 12500, 50000, 100000 or more primers that are at least 85%identical or complementary to any primer of Tables 2, 3, 13, 14, 15, 17and 19. In some embodiments, the composition includes at least one 1, 2,5, 10, 25, 50, 100, 250, 500, 750, 1000, 2500, 5000, 7500, 10000, 12500,50000, 100000 or more primers selected from Tables 2, 3, 13, 14, 15, 17and 19, or their complements, wherein at least one primer includes atleast one nucleotide substitution. A nucleotide substitution includesreplacement of any nucleotide residue or nucleobase of any primer withany other nucleotide or nucleobase, and can include, for example, purineto purine substitutions, pyrimidine to pyrimidine substitutions, purineto pyrimidine substitutions, and pyrimidine to purine substitutions. Insome embodiments, the at least one primer of the composition can includeany one, two, three, four or more nucleotide substitutions. In someembodiments, the at least one primer of the composition includes atleast one primer in which any one, some or all of the uracil-containingnucleotide residues or nucleobases of the primer are replaced with athymine-containing nucleotide residue or nucleobase. In someembodiments, the at least one primer of the composition includes atleast one primer in which any one, two, three, four, five or moreuracil-containing nucleotide residues or nucleobases of the primer arereplaced with a thymine-containing nucleotide residue or nucleobase.

In some embodiments, a target-specific primer pair can include a nucleicacid sequence including a somatic or germline mutation. In someembodiments, the germline or somatic mutation can be found in any one ormore of the genes provided in Tables 1, 4, 16 or 18. In someembodiments, the target-specific primer pairs can be used to amplify atarget sequence that can be used to detect the presence of mutations atless than 5% allele frequency. In some embodiments, the plurality oftarget-specific primers includes at least 500, at least 1000, at least3000, at least 6000, at least 10000, at least 12000, or moretarget-specific primer pairs.

In some embodiments, the disclosure relates generally to a kit forperforming multiplex nucleic acid amplification or multiplex nucleicacid synthesis. In some embodiments, the kit comprises a plurality oftarget-specific primers. In some embodiments, the kit can furtherinclude a polymerase, at least one adapter and/or a cleaving reagent. Insome embodiments, the kit can also include dATP, dCTP, dGTP, dTTP and/oran antibody. In some embodiments, the cleaving reagent is any reagentthat can cleave one or more cleaving groups present in one or moretarget-specific primers. In some embodiments the cleaving reagent caninclude an enzyme or chemical reagent. In some embodiments, the cleavingreagent can include an enzyme with an affinity for apurinic bases. Insome embodiments, the cleaving reagent can include a first enzyme withan affinity for a first cleavable group and can further include a secondenzyme with an affinity for a second cleavable group. In someembodiments, the kit can further include an enzyme with an affinity forabasic sites. In some embodiments, the polymerase is a thermostablepolymerase. In some embodiments, the kits can include one or morepreservatives, adjuvants or nucleic acid sequencing barcodes.

In some embodiments, the disclosure relates generally to methods (aswell as related compositions, systems, kits and apparatuses) fordetermining copy number variation comprising performing any of theamplification methods disclosed herein.

DETAILED DESCRIPTION

The following description of various exemplary embodiments is exemplaryand explanatory only and is not to be construed as limiting orrestrictive in any way. Other embodiments, features, objects, andadvantages of the present teachings will be apparent from thedescription and accompanying drawings, and from the claims.

As used herein, “amplify”, “amplifying” or “amplification reaction” andtheir derivatives, refer generally to any action or process whereby atleast a portion of a nucleic acid molecule (referred to as a templatenucleic acid molecule) is replicated or copied into at least oneadditional nucleic acid molecule. The additional nucleic acid moleculeoptionally includes sequence that is substantially identical orsubstantially complementary to at least some portion of the templatenucleic acid molecule. The template nucleic acid molecule can besingle-stranded or double-stranded and the additional nucleic acidmolecule can independently be single-stranded or double-stranded. Insome embodiments, amplification includes a template-dependent in vitroenzyme-catalyzed reaction for the production of at least one copy of atleast some portion of the nucleic acid molecule or the production of atleast one copy of a nucleic acid sequence that is complementary to atleast some portion of the nucleic acid molecule. Amplificationoptionally includes linear or exponential replication of a nucleic acidmolecule. In some embodiments, such amplification is performed usingisothermal conditions; in other embodiments, such amplification caninclude thermocycling. In some embodiments, the amplification is amultiplex amplification that includes the simultaneous amplification ofa plurality of target sequences in a single amplification reaction. Atleast some of the target sequences can be situated on the same nucleicacid molecule or on different target nucleic acid molecules included inthe single amplification reaction. In some embodiments, “amplification”includes amplification of at least some portion of DNA- and RNA-basednucleic acids alone, or in combination. The amplification reaction caninclude single or double-stranded nucleic acid substrates and canfurther including any of the amplification processes known to one ofordinary skill in the art. In some embodiments, the amplificationreaction includes polymerase chain reaction (PCR).

As used herein, “amplification conditions” and its derivatives,generally refers to conditions suitable for amplifying one or morenucleic acid sequences. Such amplification can be linear or exponential.In some embodiments, the amplification conditions can include isothermalconditions or alternatively can include thermocyling conditions, or acombination of isothermal and themocycling conditions. In someembodiments, the conditions suitable for amplifying one or more nucleicacid sequences includes polymerase chain reaction (PCR) conditions.Typically, the amplification conditions refer to a reaction mixture thatis sufficient to amplify nucleic acids such as one or more targetsequences, or to amplify an amplified target sequence ligated to one ormore adapters, e.g., an adapter-ligated amplified target sequence.Generally, the amplification conditions include a catalyst foramplification or for nucleic acid synthesis, for example a polymerase; aprimer that possesses some degree of complementarity to the nucleic acidto be amplified; and nucleotides, such as deoxyribonucleotidetriphosphates (dNTPs) to promote extension of the primer once hybridizedto the nucleic acid. The amplification conditions can requirehybridization or annealing of a primer to a nucleic acid, extension ofthe primer and a denaturing step in which the extended primer isseparated from the nucleic acid sequence undergoing amplification.Typically, but not necessarily, amplification conditions can includethermocycling; in some embodiments, amplification conditions include aplurality of cycles where the steps of annealing, extending andseparating are repeated. Typically, the amplification conditions includecations such as Mg⁺⁺ or Mn⁺⁺ (e.g., MgCl₂, etc) and can also includevarious modifiers of ionic strength.

As used herein, “target sequence” or “target sequence of interest” andits derivatives, refers generally to any single or double-strandednucleic acid sequence that can be amplified or synthesized according tothe disclosure, including any nucleic acid sequence suspected orexpected to be present in a sample. In some embodiments, the targetsequence is present in double-stranded form and includes at least aportion of the particular nucleotide sequence to be amplified orsynthesized, or its complement, prior to the addition of target-specificprimers or appended adapters. Target sequences can include the nucleicacids to which primers useful in the amplification or synthesis reactioncan hybridize prior to extension by a polymerase. In some embodiments,the term refers to a nucleic acid sequence whose sequence identity,ordering or location of nucleotides is determined by one or more of themethods of the disclosure.

As defined herein, “sample” and its derivatives, is used in its broadestsense and includes any specimen, culture and the like that is suspectedof including a target. In some embodiments, the sample comprises DNA,RNA, PNA, LNA, chimeric, hybrid, or multiplex-forms of nucleic acids.The sample can include any biological, clinical, surgical, agricultural,atmospheric or aquatic-based specimen containing one or more nucleicacids. The term also includes any isolated nucleic acid sample such agenomic DNA, fresh-frozen or formalin-fixed paraffin-embedded nucleicacid specimen.

As used herein, “contacting” and its derivatives, when used in referenceto two or more components, refers generally to any process whereby theapproach, proximity, mixture or commingling of the referenced componentsis promoted or achieved without necessarily requiring physical contactof such components, and includes mixing of solutions containing any oneor more of the referenced components with each other. The referencedcomponents may be contacted in any particular order or combination andthe particular order of recitation of components is not limiting. Forexample, “contacting A with B and C” encompasses embodiments where A isfirst contacted with B then C, as well as embodiments where C iscontacted with A then B, as well as embodiments where a mixture of A andC is contacted with B, and the like. Furthermore, such contacting doesnot necessarily require that the end result of the contacting process bea mixture including all of the referenced components, as long as at somepoint during the contacting process all of the referenced components aresimultaneously present or simultaneously included in the same mixture orsolution. For example, “contacting A with B and C” can includeembodiments wherein C is first contacted with A to form a first mixture,which first mixture is then contacted with B to form a second mixture,following which C is removed from the second mixture; optionally A canthen also be removed, leaving only B. Where one or more of thereferenced components to be contacted includes a plurality (e.g,“contacting a target sequence with a plurality of target-specificprimers and a polymerase”), then each member of the plurality can beviewed as an individual component of the contacting process, such thatthe contacting can include contacting of any one or more members of theplurality with any other member of the plurality and/or with any otherreferenced component (e.g., some but not all of the plurality of targetspecific primers can be contacted with a target sequence, then apolymerase, and then with other members of the plurality oftarget-specific primers) in any order or combination.

As used herein, the term “primer” and its derivatives refer generally toany polynucleotide that can hybridize to a target sequence of interest.In some embodiments, the primer can also serve to prime nucleic acidsynthesis. Typically, the primer functions as a substrate onto whichnucleotides can be polymerized by a polymerase; in some embodiments,however, the primer can become incorporated into the synthesized nucleicacid strand and provide a site to which another primer can hybridize toprime synthesis of a new strand that is complementary to the synthesizednucleic acid molecule. The primer may be comprised of any combination ofnucleotides or analogs thereof, which may be optionally linked to form alinear polymer of any suitable length. In some embodiments, the primeris a single-stranded oligonucleotide or polynucleotide. (For purposes ofthis disclosure, the terms ‘polynucleotide” and “oligonucleotide” areused interchangeably herein and do not necessarily indicate anydifference in length between the two). In some embodiments, the primeris single-stranded but it can also be double-stranded. The primeroptionally occurs naturally, as in a purified restriction digest, or canbe produced synthetically. In some embodiments, the primer acts as apoint of initiation for amplification or synthesis when exposed toamplification or synthesis conditions; such amplification or synthesiscan occur in a template-dependent fashion and optionally results information of a primer extension product that is complementary to atleast a portion of the target sequence. Exemplary amplification orsynthesis conditions can include contacting the primer with apolynucleotide template (e.g., a template including a target sequence),nucleotides and an inducing agent such as a polymerase at a suitabletemperature and pH to induce polymerization of nucleotides onto an endof the target-specific primer. If double-stranded, the primer canoptionally be treated to separate its strands before being used toprepare primer extension products. In some embodiments, the primer is anoligodeoxyribonucleotide or an oligoribonucleotide. In some embodiments,the primer can include one or more nucleotide analogs. The exact lengthand/or composition, including sequence, of the target-specific primercan influence many properties, including melting temperature (Tm), GCcontent, formation of secondary structures, repeat nucleotide motifs,length of predicted primer extension products, extent of coverage acrossa nucleic acid molecule of interest, number of primers present in asingle amplification or synthesis reaction, presence of nucleotideanalogs or modified nucleotides within the primers, and the like. Insome embodiments, a primer can be paired with a compatible primer withinan amplification or synthesis reaction to form a primer pair consistingor a forward primer and a reverse primer. In some embodiments, theforward primer of the primer pair includes a sequence that issubstantially complementary to at least a portion of a strand of anucleic acid molecule, and the reverse primer of the primer of theprimer pair includes a sequence that is substantially identical to atleast of portion of the strand. In some embodiments, the forward primerand the reverse primer are capable of hybridizing to opposite strands ofa nucleic acid duplex. Optionally, the forward primer primes synthesisof a first nucleic acid strand, and the reverse primer primes synthesisof a second nucleic acid strand, wherein the first and second strandsare substantially complementary to each other, or can hybridize to forma double-stranded nucleic acid molecule. In some embodiments, one end ofan amplification or synthesis product is defined by the forward primerand the other end of the amplification or synthesis product is definedby the reverse primer. In some embodiments, where the amplification orsynthesis of lengthy primer extension products is required, such asamplifying an exon, coding region, or gene, several primer pairs can becreated than span the desired length to enable sufficient amplificationof the region. In some embodiments, a primer can include one or morecleavable groups. In some embodiments, primer lengths are in the rangeof about 10 to about 60 nucleotides, about 12 to about 50 nucleotidesand about 15 to about 40 nucleotides in length. Typically, a primer iscapable of hybridizing to a corresponding target sequence and undergoingprimer extension when exposed to amplification conditions in thepresence of dNTPS and a polymerase. In some instances, the particularnucleotide sequence or a portion of the primer is known at the outset ofthe amplification reaction or can be determined by one or more of themethods disclosed herein. In some embodiments, the primer includes oneor more cleavable groups at one or more locations within the primer.

As used herein, “target-specific primer” and its derivatives, refersgenerally to a single stranded or double-stranded polynucleotide,typically an oligonucleotide, that includes at least one sequence thatis at least 50% complementary, typically at least 75% complementary orat least 85% complementary, more typically at least 90% complementary,more typically at least 95% complementary, more typically at least 98%or at least 99% complementary, or identical, to at least a portion of anucleic acid molecule that includes a target sequence. In suchinstances, the target-specific primer and target sequence are describedas “corresponding” to each other. In some embodiments, thetarget-specific primer is capable of hybridizing to at least a portionof its corresponding target sequence (or to a complement of the targetsequence); such hybridization can optionally be performed under standardhybridization conditions or under stringent hybridization conditions. Insome embodiments, the target-specific primer is not capable ofhybridizing to the target sequence, or to its complement, but is capableof hybridizing to a portion of a nucleic acid strand including thetarget sequence, or to its complement. In some embodiments, thetarget-specific primer includes at least one sequence that is at least75% complementary, typically at least 85% complementary, more typicallyat least 90% complementary, more typically at least 95% complementary,more typically at least 98% complementary, or more typically at least99% complementary, to at least a portion of the target sequence itself;in other embodiments, the target-specific primer includes at least onesequence that is at least 75% complementary, typically at least 85%complementary, more typically at least 90% complementary, more typicallyat least 95% complementary, more typically at least 98% complementary,or more typically at least 99% complementary, to at least a portion ofthe nucleic acid molecule other than the target sequence. In someembodiments, the target-specific primer is substantiallynon-complementary to other target sequences present in the sample;optionally, the target-specific primer is substantiallynon-complementary to other nucleic acid molecules present in the sample.In some embodiments, nucleic acid molecules present in the sample thatdo not include or correspond to a target sequence (or to a complement ofthe target sequence) are referred to as “non-specific” sequences or“non-specific nucleic acids”. In some embodiments, the target-specificprimer is designed to include a nucleotide sequence that issubstantially complementary to at least a portion of its correspondingtarget sequence. In some embodiments, a target-specific primer is atleast 95% complementary, or at least 99% complementary, or identical,across its entire length to at least a portion of a nucleic acidmolecule that includes its corresponding target sequence. In someembodiments, a target-specific primer can be at least 90%, at least 95%complementary, at least 98% complementary or at least 99% complementary,or identical, across its entire length to at least a portion of itscorresponding target sequence. In some embodiments, a forwardtarget-specific primer and a reverse target-specific primer define atarget-specific primer pair that can be used to amplify the targetsequence via template-dependent primer extension. Typically, each primerof a target-specific primer pair includes at least one sequence that issubstantially complementary to at least a portion of a nucleic acidmolecule including a corresponding target sequence but that is less than50% complementary to at least one other target sequence in the sample.In some embodiments, amplification can be performed using multipletarget-specific primer pairs in a single amplification reaction, whereineach primer pair includes a forward target-specific primer and a reversetarget-specific primer, each including at least one sequence thatsubstantially complementary or substantially identical to acorresponding target sequence in the sample, and each primer pair havinga different corresponding target sequence. In some embodiments, thetarget-specific primer can be substantially non-complementary at its 3′end or its 5′ end to any other target-specific primer present in anamplification reaction. In some embodiments, the target-specific primercan include minimal cross hybridization to other target-specific primersin the amplification reaction. In some embodiments, target-specificprimers include minimal cross-hybridization to non-specific sequences inthe amplification reaction mixture. In some embodiments, thetarget-specific primers include minimal self-complementarity. In someembodiments, the target-specific primers can include one or morecleavable groups located at the 3′ end. In some embodiments, thetarget-specific primers can include one or more cleavable groups locatednear or about a central nucleotide of the target-specific primer. Insome embodiments, one of more targets-specific primers includes onlynon-cleavable nucleotides at the 5′ end of the target-specific primer.In some embodiments, a target specific primer includes minimalnucleotide sequence overlap at the 3′ end or the 5′ end of the primer ascompared to one or more different target-specific primers, optionally inthe same amplification reaction. In some embodiments 1, 2, 3, 4, 5, 6,7, 8, 9, 10 or more, target-specific primers in a single reactionmixture include one or more of the above embodiments. In someembodiments, substantially all of the plurality of target-specificprimers in a single reaction mixture includes one or more of the aboveembodiments.

As used herein, “polymerase” and its derivatives, generally refers toany enzyme that can catalyze the polymerization of nucleotides(including analogs thereof) into a nucleic acid strand. Typically butnot necessarily, such nucleotide polymerization can occur in atemplate-dependent fashion. Such polymerases can include withoutlimitation naturally occurring polymerases and any subunits andtruncations thereof, mutant polymerases, variant polymerases,recombinant, fusion or otherwise engineered polymerases, chemicallymodified polymerases, synthetic molecules or assemblies, and anyanalogs, derivatives or fragments thereof that retain the ability tocatalyze such polymerization. Optionally, the polymerase can be a mutantpolymerase comprising one or more mutations involving the replacement ofone or more amino acids with other amino acids, the insertion ordeletion of one or more amino acids from the polymerase, or the linkageof parts of two or more polymerases. Typically, the polymerase comprisesone or more active sites at which nucleotide binding and/or catalysis ofnucleotide polymerization can occur. Some exemplary polymerases includewithout limitation DNA polymerases and RNA polymerases. The term“polymerase” and its variants, as used herein, also refers to fusionproteins comprising at least two portions linked to each other, wherethe first portion comprises a peptide that can catalyze thepolymerization of nucleotides into a nucleic acid strand and is linkedto a second portion that comprises a second polypeptide. In someembodiments, the second polypeptide can include a reporter enzyme or aprocessivity-enhancing domain. Optionally, the polymerase can possess 5′exonuclease activity or terminal transferase activity. In someembodiments, the polymerase can be optionally reactivated, for examplethrough the use of heat, chemicals or re-addition of new amounts ofpolymerase into a reaction mixture. In some embodiments, the polymerasecan include a hot-start polymerase or an aptamer based polymerase thatoptionally can be reactivated.

As used herein, the term “nucleotide” and its variants comprises anycompound, including without limitation any naturally occurringnucleotide or analog thereof, which can bind selectively to, or can bepolymerized by, a polymerase. Typically, but not necessarily, selectivebinding of the nucleotide to the polymerase is followed bypolymerization of the nucleotide into a nucleic acid strand by thepolymerase; occasionally however the nucleotide may dissociate from thepolymerase without becoming incorporated into the nucleic acid strand,an event referred to herein as a “non-productive” event. Suchnucleotides include not only naturally occurring nucleotides but alsoany analogs, regardless of their structure, that can bind selectivelyto, or can be polymerized by, a polymerase. While naturally occurringnucleotides typically comprise base, sugar and phosphate moieties, thenucleotides of the present disclosure can include compounds lacking anyone, some or all of such moieties. In some embodiments, the nucleotidecan optionally include a chain of phosphorus atoms comprising three,four, five, six, seven, eight, nine, ten or more phosphorus atoms. Insome embodiments, the phosphorus chain can be attached to any carbon ofa sugar ring, such as the 5′ carbon. The phosphorus chain can be linkedto the sugar with an intervening O or S. In one embodiment, one or morephosphorus atoms in the chain can be part of a phosphate group having Pand O. In another embodiment, the phosphorus atoms in the chain can belinked together with intervening O, NH, S, methylene, substitutedmethylene, ethylene, substituted ethylene, CNH₂, C(O), C(CH₂), CH₂CH₂,or C(OH)CH₂R (where R can be a 4-pyridine or 1-imidazole). In oneembodiment, the phosphorus atoms in the chain can have side groupshaving O, BH₃, or S. In the phosphorus chain, a phosphorus atom with aside group other than O can be a substituted phosphate group. In thephosphorus chain, phosphorus atoms with an intervening atom other than Ocan be a substituted phosphate group. Some examples of nucleotideanalogs are described in Xu, U.S. Pat. No. 7,405,281. In someembodiments, the nucleotide comprises a label and referred to herein asa “labeled nucleotide”; the label of the labeled nucleotide is referredto herein as a “nucleotide label”. In some embodiments, the label can bein the form of a fluorescent dye attached to the terminal phosphategroup, i.e., the phosphate group most distal from the sugar. Someexamples of nucleotides that can be used in the disclosed methods andcompositions include, but are not limited to, ribonucleotides,deoxyribonucleotides, modified ribonucleotides, modifieddeoxyribonucleotides, ribonucleotide polyphosphates, deoxyribonucleotidepolyphosphates, modified ribonucleotide polyphosphates, modifieddeoxyribonucleotide polyphosphates, peptide nucleotides, modifiedpeptide nucleotides, metallonucleosides, phosphonate nucleosides, andmodified phosphate-sugar backbone nucleotides, analogs, derivatives, orvariants of the foregoing compounds, and the like. In some embodiments,the nucleotide can comprise non-oxygen moieties such as, for example,thio- or borano-moieties, in place of the oxygen moiety bridging thealpha phosphate and the sugar of the nucleotide, or the alpha and betaphosphates of the nucleotide, or the beta and gamma phosphates of thenucleotide, or between any other two phosphates of the nucleotide, orany combination thereof. “Nucleotide 5′-triphosphate” refers to anucleotide with a triphosphate ester group at the 5′ position, and aresometimes denoted as “NTP”, or “dNTP” and “ddNTP” to particularly pointout the structural features of the ribose sugar. The triphosphate estergroup can include sulfur substitutions for the various oxygens, e.g..alpha.-thio-nucleotide 5′-triphosphates. For a review of nucleic acidchemistry, see: Shabarova, Z. and Bogdanov, A. Advanced OrganicChemistry of Nucleic Acids, VCH, New York, 1994.

The term “extension” and its variants, as used herein, when used inreference to a given primer, comprises any in vivo or in vitro enzymaticactivity characteristic of a given polymerase that relates topolymerization of one or more nucleotides onto an end of an existingnucleic acid molecule. Typically but not necessarily such primerextension occurs in a template-dependent fashion; duringtemplate-dependent extension, the order and selection of bases is drivenby established base pairing rules, which can include Watson-Crick typebase pairing rules or alternatively (and especially in the case ofextension reactions involving nucleotide analogs) by some other type ofbase pairing paradigm. In one non-limiting example, extension occurs viapolymerization of nucleotides on the 3′OH end of the nucleic acidmolecule by the polymerase.

The term “portion” and its variants, as used herein, when used inreference to a given nucleic acid molecule, for example a primer or atemplate nucleic acid molecule, comprises any number of contiguousnucleotides within the length of the nucleic acid molecule, includingthe partial or entire length of the nucleic acid molecule.

The terms “identity” and “identical” and their variants, as used herein,when used in reference to two or more nucleic acid sequences, refer tosimilarity in sequence of the two or more sequences (e.g., nucleotide orpolypeptide sequences). In the context of two or more homologoussequences, the percent identity or homology of the sequences orsubsequences thereof indicates the percentage of all monomeric units(e.g., nucleotides or amino acids) that are the same (i.e., about 70%identity, preferably 75%, 80%, 85%, 90%, 95%, 98% or 99% identity). Thepercent identity can be over a specified region, when compared andaligned for maximum correspondence over a comparison window, ordesignated region as measured using a BLAST or BLAST 2.0 sequencecomparison algorithms with default parameters described below, or bymanual alignment and visual inspection. Sequences are said to be“substantially identical” when there is at least 85% identity at theamino acid level or at the nucleotide level. Preferably, the identityexists over a region that is at least about 25, 50, or 100 residues inlength, or across the entire length of at least one compared sequence. Atypical algorithm for determining percent sequence identity and sequencesimilarity are the BLAST and BLAST 2.0 algorithms, which are describedin Altschul et al, Nuc. Acids Res. 25:3389-3402 (1977). Other methodsinclude the algorithms of Smith & Waterman, Adv. Appl. Math. 2:482(1981), and Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), etc.Another indication that two nucleic acid sequences are substantiallyidentical is that the two molecules or their complements hybridize toeach other under stringent hybridization conditions.

The terms “complementary” and “complement” and their variants, as usedherein, refer to any two or more nucleic acid sequences (e.g., portionsor entireties of template nucleic acid molecules, target sequencesand/or primers) that can undergo cumulative base pairing at two or moreindividual corresponding positions in antiparallel orientation, as in ahybridized duplex. Such base pairing can proceed according to any set ofestablished rules, for example according to Watson-Crick base pairingrules or according, to some other base pairing paradigm. Optionallythere can be “complete” or “total” complementarity between a first andsecond nucleic acid sequence where each nucleotide in the first nucleicacid sequence can undergo a stabilizing base pairing interaction with anucleotide in the corresponding antiparallel position on the secondnucleic acid sequence. “Partial” complementarity describes nucleic acidsequences in which at least 20%, but less than 100%, of the residues ofone nucleic acid sequence are complementary to residues in the othernucleic acid sequence. In some embodiments, at least 50%, but less than100%, of the residues of one nucleic acid sequence are complementary toresidues in the other nucleic acid sequence. In some embodiments, atleast 70%, 80%, 90%, 95% or 98%, but less than 100%, of the residues ofone nucleic acid sequence are complementary to residues in the othernucleic acid sequence. Sequences are said to be “substantiallycomplementary” when at least 85% of the residues of one nucleic acidsequence are complementary to residues in the other nucleic acidsequence. In some embodiments, two complementary or substantiallycomplementary sequences are capable of hybridizing to each other understandard or stringent hybridization conditions. “Non-complementary”describes nucleic acid sequences in which less than 20% of the residuesof one nucleic acid sequence are complementary to residues in the othernucleic acid sequence. Sequences are said to be “substantiallynon-complementary” when less than 15% of the residues of one nucleicacid sequence are complementary to residues in the other nucleic acidsequence. In some embodiments, two non-complementary or substantiallynon-complementary sequences cannot hybridize to each other understandard or stringent hybridization conditions. A “mismatch” is presentat any position in the two opposed nucleotides are not complementary.Complementary nucleotides include nucleotides that are efficientlyincorporated by DNA polymerases opposite each other during DNAreplication under physiological conditions. In a typical embodiment,complementary nucleotides can form base pairs with each other, such asthe A-T/U and G-C base pairs formed through specific Watson-Crick typehydrogen bonding, or base pairs formed through some other type of basepairing paradigm, between the nucleobases of nucleotides and/orpolynucleotides in positions antiparallel to each other. Thecomplementarity of other artificial base pairs can be based on othertypes of hydrogen bonding and/or hydrophobicity of bases and/or shapecomplementarity between bases.

As used herein, “amplified target sequences” and its derivatives, refersgenerally to a nucleic acid sequence produced by the amplificationof/amplifying the target sequences using target-specific primers and themethods provided herein. The amplified target sequences may be either ofthe same sense (the positive strand produced in the second round andsubsequent even-numbered rounds of amplification) or antisense (i.e.,the negative strand produced during the first and subsequentodd-numbered rounds of amplification) with respect to the targetsequences. For the purposes of this disclosure, the amplified targetsequences are typically less than 50% complementary to any portion ofanother amplified target sequence in the reaction.

As used herein, the terms “ligating”, “ligation” and their derivativesrefer generally to the act or process for covalently linking two or moremolecules together, for example, covalently linking two or more nucleicacid molecules to each other. In some embodiments, ligation includesjoining nicks between adjacent nucleotides of nucleic acids. In someembodiments, ligation includes forming a covalent bond between an end ofa first and an end of a second nucleic acid molecule. In someembodiments, for example embodiments wherein the nucleic acid moleculesto be ligated include conventional nucleotide residues, the ligation caninclude forming a covalent bond between a 5′ phosphate group of onenucleic acid and a 3′ hydroxyl group of a second nucleic acid therebyforming a ligated nucleic acid molecule. In some embodiments, any meansfor joining nicks or bonding a 5′ phosphate to a 3′ hydroxyl betweenadjacent nucleotides can be employed. In an exemplary embodiment, anenzyme such as a ligase can be used. Generally for the purposes of thisdisclosure, an amplified target sequence can be ligated to an adapter togenerate an adapter-ligated amplified target sequence.

As used herein, “ligase” and its derivatives, refers generally to anyagent capable of catalyzing the ligation of two substrate molecules. Insome embodiments, the ligase includes an enzyme capable of catalyzingthe joining of nicks between adjacent nucleotides of a nucleic acid. Insome embodiments, the ligase includes an enzyme capable of catalyzingthe formation of a covalent bond between a 5′ phosphate of one nucleicacid molecule to a 3′ hydroxyl of another nucleic acid molecule therebyforming a ligated nucleic acid molecule. Suitable ligases may include,but not limited to, T4 DNA ligase, T4 RNA ligase, and E. coli DNAligase.

As used herein, “ligation conditions” and its derivatives, generallyrefers to conditions suitable for ligating two molecules to each other.In some embodiments, the ligation conditions are suitable for sealingnicks or gaps between nucleic acids. As defined herein, a “nick” or“gap” refers to a nucleic acid molecule that lacks a directly bound 5′phosphate of a mononucleotide pentose ring to a 3′ hydroxyl of aneighboring mononucleotide pentose ring within internal nucleotides of anucleic acid sequence. As used herein, the term nick or gap isconsistent with the use of the term in the art. Typically, a nick or gapcan be ligated in the presence of an enzyme, such as ligase at anappropriate temperature and pH. In some embodiments, T4 DNA ligase canjoin a nick between nucleic acids at a temperature of about 70-72° C.

As used herein, “blunt-end ligation” and its derivatives, refersgenerally to ligation of two blunt-end double-stranded nucleic acidmolecules to each other. A “blunt end” refers to an end of adouble-stranded nucleic acid molecule wherein substantially all of thenucleotides in the end of one strand of the nucleic acid molecule arebase paired with opposing nucleotides in the other strand of the samenucleic acid molecule. A nucleic acid molecule is not blunt ended if ithas an end that includes a single-stranded portion greater than twonucleotides in length, referred to herein as an “overhang”. In someembodiments, the end of nucleic acid molecule does not include anysingle stranded portion, such that every nucleotide in one strand of theend is based paired with opposing nucleotides in the other strand of thesame nucleic acid molecule. In some embodiments, the ends of the twoblunt ended nucleic acid molecules that become ligated to each other donot include any overlapping, shared or complementary sequence.Typically, blunted-end ligation excludes the use of additionaloligonucleotide adapters to assist in the ligation of thedouble-stranded amplified target sequence to the double-strandedadapter, such as patch oligonucleotides as described in Mitra andVarley, US2010/0129874, published May 27, 2010. In some embodiments,blunt-ended ligation includes a nick translation reaction to seal a nickcreated during the ligation process.

As used herein, the terms “adapter” or “adapter and its complements” andtheir derivatives, refers generally to any linear oligonucleotide whichcan be ligated to a nucleic acid molecule of the disclosure. Optionally,the adapter includes a nucleic acid sequence that is not substantiallycomplementary to the 3′ end or the 5′ end of at least one targetsequences within the sample. In some embodiments, the adapter issubstantially non-complementary to the 3′ end or the 5′ end of anytarget sequence present in the sample. In some embodiments, the adapterincludes any single stranded or double-stranded linear oligonucleotidethat is not substantially complementary to an amplified target sequence.In some embodiments, the adapter is substantially non-complementary toat least one, some or all of the nucleic acid molecules of the sample.In some embodiments, suitable adapter lengths are in the range of about10-100 nucleotides, about 12-60 nucleotides and about 15-50 nucleotidesin length. Generally, the adapter can include any combination ofnucleotides and/or nucleic acids. In some aspects, the adapter caninclude one or more cleavable groups at one or more locations. Inanother aspect, the adapter can include a sequence that is substantiallyidentical, or substantially complementary, to at least a portion of aprimer, for example a universal primer. In some embodiments, the adaptercan include a barcode or tag to assist with downstream cataloguing,identification or sequencing. In some embodiments, a single-strandedadapter can act as a substrate for amplification when ligated to anamplified target sequence, particularly in the presence of a polymeraseand dNTPs under suitable temperature and pH.

As used herein, “reamplifying” or “reamplification” and theirderivatives refer generally to any process whereby at least a portion ofan amplified nucleic acid molecule is further amplified via any suitableamplification process (referred to in some embodiments as a “secondary”amplification or “reamplification”, thereby producing a reamplifiednucleic acid molecule. The secondary amplification need not be identicalto the original amplification process whereby the amplified nucleic acidmolecule was produced; nor need the reamplified nucleic acid molecule becompletely identical or completely complementary to the amplifiednucleic acid molecule; all that is required is that the reamplifiednucleic acid molecule include at least a portion of the amplifiednucleic acid molecule or its complement. For example, thereamplification can involve the use of different amplificationconditions and/or different primers, including different target-specificprimers than the primary amplification.

As defined herein, a “cleavable group” generally refers to any moietythat once incorporated into a nucleic acid can be cleaved underappropriate conditions. For example, a cleavable group can beincorporated into a target-specific primer, an amplified sequence, anadapter or a nucleic acid molecule of the sample. In an exemplaryembodiment, a target-specific primer can include a cleavable group thatbecomes incorporated into the amplified product and is subsequentlycleaved after amplification, thereby removing a portion, or all, of thetarget-specific primer from the amplified product. The cleavable groupcan be cleaved or otherwise removed from a target-specific primer, anamplified sequence, an adapter or a nucleic acid molecule of the sampleby any acceptable means. For example, a cleavable group can be removedfrom a target-specific primer, an amplified sequence, an adapter or anucleic acid molecule of the sample by enzymatic, thermal,photo-oxidative or chemical treatment. In one aspect, a cleavable groupcan include a nucleobase that is not naturally occurring. For example,an oligodeoxyribonucleotide can include one or more RNA nucleobases,such as uracil that can be removed by a uracil glycosylase. In someembodiments, a cleavable group can include one or more modifiednucleobases (such as 7-methylguanine, 8-oxo-guanine, xanthine,hypoxanthine, 5,6-dihydrouracil or 5-methylcytosine) or one or moremodified nucleosides (i.e., 7-methylguanosine, 8-oxo-deoxyguanosine,xanthosine, inosine, dihydrouridine or 5-methylcytidine). The modifiednucleobases or nucleotides can be removed from the nucleic acid byenzymatic, chemical or thermal means. In one embodiment, a cleavablegroup can include a moiety that can be removed from a primer afteramplification (or synthesis) upon exposure to ultraviolet light (i.e.,bromodeoxyuridine). In another embodiment, a cleavable group can includemethylated cytosine. Typically, methylated cytosine can be cleaved froma primer for example, after induction of amplification (or synthesis),upon sodium bisulfite treatment. In some embodiments, a cleavable moietycan include a restriction site. For example, a primer or target sequencecan include a nucleic acid sequence that is specific to one or morerestriction enzymes, and following amplification (or synthesis), theprimer or target sequence can be treated with the one or morerestriction enzymes such that the cleavable group is removed. Typically,one or more cleavable groups can be included at one or more locationswith a target-specific primer, an amplified sequence, an adapter or anucleic acid molecule of the sample.

As used herein, “cleavage step” and its derivatives, generally refers toany process by which a cleavable group is cleaved or otherwise removedfrom a target-specific primer, an amplified sequence, an adapter or anucleic acid molecule of the sample. In some embodiments, the cleavagesteps involves a chemical, thermal, photo-oxidative or digestiveprocess.

As used herein, the term “hybridization” is consistent with its use inthe art, and generally refers to the process whereby two nucleic acidmolecules undergo base pairing interactions. Two nucleic acid moleculemolecules are said to be hybridized when any portion of one nucleic acidmolecule is base paired with any portion of the other nucleic acidmolecule; it is not necessarily required that the two nucleic acidmolecules be hybridized across their entire respective lengths and insome embodiments, at least one of the nucleic acid molecules can includeportions that are not hybridized to the other nucleic acid molecule. Thephrase “hybridizing under stringent conditions” and its variants refersgenerally to conditions under which hybridization of a target-specificprimer to a target sequence occurs in the presence of high hybridizationtemperature and low ionic strength. In one, exemplary embodiment,stringent hybridization conditions include an aqueous environmentcontaining about 30 mM magnesium sulfate, about 300 mM Tris-sulfate atpH 8.9, and about 90 mM ammonium sulfate at about 60-68° C., orequivalents thereof. As used herein, the phrase “standard hybridizationconditions” and its variants refers generally to conditions under whichhybridization of a primer to an oligonucleotide (i.e., a targetsequence), occurs in the presence of low hybridization temperature andhigh ionic strength. In one exemplary embodiment, standard hybridizationconditions include an aqueous environment containing about 100 mMmagnesium sulfate, about 500 mM Tris-sulfate at pH 8.9, and about 200 mMammonium sulfate at about 50-55° C., or equivalents thereof.

As used herein, “triple nucleotide motif” and its derivatives, refersgenerally to any nucleotide sequence that is repeated contiguously overthree nucleotides e.g., AAA or CCC. Generally, a triple nucleotide motifis not repeated more than five times in a target-specific primer (oradapter) of the disclosure.

As used herein, “an ACA nucleotide motif” and its derivatives, refersgenerally to the nucleotide sequence “ACA”. Generally, this motif is notrepeated three or more times in a target-specific primer (or adapter) ofthe disclosure.

As used herein, “homopolymer” and its derivatives, refers generally toany repeating nucleotide sequence that is eight nucleotides or greaterin length e.g., AAAAAAAA or CCCCCCCC. Generally, a homopolymer asdefined herein is not present in a target-specific primer (or adapter)of the disclosure.

As used herein, “GC content” and its derivatives, refers generally tothe cytosine and guanine content of a nucleic acid molecule. Generally,the GC content of a target-specific primer (or adapter) of thedisclosure is 85% or lower. More typically, the GC content of atarget-specific primer or adapter of the disclosure is between 15-85%.

As used herein, the term “end” and its variants, when used in referenceto a nucleic acid molecule, for example a target sequence or amplifiedtarget sequence, can include the terminal 30 nucleotides, the terminal20 and even more typically the terminal 15 nucleotides of the nucleicacid molecule. A linear nucleic acid molecule comprised of linked seriesof contiguous nucleotides typically includes at least two ends. In someembodiments, one end of the nucleic acid molecule can include a 3′hydroxyl group or its equivalent, and can be referred to as the “3′ end”and its derivatives. Optionally, the 3′ end includes a 3′ hydroxyl groupthat is not linked to a 5′ phosphate group of a mononucleotide pentosering. Typically, the 3′ end includes one or more 5′ linked nucleotideslocated adjacent to the nucleotide including the unlinked 3′ hydroxylgroup, typically the 30 nucleotides located adjacent to the 3′ hydroxyl,typically the terminal 20 and even more typically the terminal 15nucleotides. Generally, the one or more linked nucleotides can berepresented as a percentage of the nucleotides present in theoligonucleotide or can be provided as a number of linked nucleotidesadjacent to the unlinked 3′ hydroxyl. For example, the 3′ end caninclude less than 50% of the nucleotide length of the oligonucleotide.In some embodiments, the 3′ end does not include any unlinked 3′hydroxyl group but can include any moiety capable of serving as a sitefor attachment of nucleotides via primer extension and/or nucleotidepolymerization. In some embodiments, the term “3′ end” for example whenreferring to a target-specific primer, can include the terminal 10nucleotides, the terminal 5 nucleotides, the terminal 4, 3, 2 or fewernucleotides at the 3′ end. In some embodiments, the term “3′ end” whenreferring to a target-specific primer can include nucleotides located atnucleotide positions 10 or fewer from the 3′ terminus.

As used herein, “5′ end”, and its derivatives, generally refers to anend of a nucleic acid molecule, for example a target sequence oramplified target sequence, which includes a free 5′ phosphate group orits equivalent. In some embodiments, the 5′ end includes a 5′ phosphategroup that is not linked to a 3′ hydroxyl of a neighboringmononucleotide pentose ring. Typically, the 5′ end includes to one ormore linked nucleotides located adjacent to the 5′ phosphate, typicallythe 30 nucleotides located adjacent to the nucleotide including the 5′phosphate group, typically the terminal 20 and even more typically theterminal 15 nucleotides. Generally, the one or more linked nucleotidescan be represented as a percentage of the nucleotides present in theoligonucleotide or can be provided as a number of linked nucleotidesadjacent to the 5′ phosphate. For example, the 5′ end can be less than50% of the nucleotide length of an oligonucleotide. In another exemplaryembodiment, the 5′ end can include about 15 nucleotides adjacent to thenucleotide including the terminal 5′ phosphate. In some embodiments, the5′ end does not include any unlinked 5′ phosphate group but can includeany moiety capable of serving as a site of attachment to a a 3′ hydroxylgroup, or to the 3′ end of another nucleic acid molecule. In someembodiments, the term “5′ end” for example when referring to atarget-specific primer, can include the terminal 10 nucleotides, theterminal 5 nucleotides, the terminal 4, 3, 2 or fewer nucleotides at the5′ end. In some embodiments, the term “5′ end” when referring to atarget-specific primer can include nucleotides located at positions 10or fewer from the 5′ terminus. In some embodiments, the 5′ end of atarget-specific primer can include only non-cleavable nucleotides, forexample nucleotides that do not contain one or more cleavable groups asdisclosed herein, or a cleavable nucleotide as would be readilydetermined by one of ordinary skill in the art.

As used herein, “protecting group” and its derivatives, refers generallyto any moiety that can be incorporated into an adapter ortarget-specific primer that imparts chemical selectivity or protects thetarget-specific primer or adapter from digestion or chemicaldegradation. Typically, but not necessarily, a protecting group caninclude modification of an existing functional group in thetarget-specific primer r adapter to achieve chemical selectivity.Suitable types of protecting groups include alcohol, amine, phosphate,carbonyl, or carboxylic acid protecting groups. In an exemplaryembodiment, the protecting group can include a spacer compound having achain of carbon atoms.

As used herein, “DNA barcode” or “DNA tagging sequence” and itsderivatives, refers generally to a unique short (6-14 nucleotide)nucleic acid sequence within an adapter that can act as a ‘key’ todistinguish or separate a plurality of amplified target sequences in asample. For the purposes of this disclosure, a DNA barcode or DNAtagging sequence can be incorporated into the nucleotide sequence of anadapter.

As used herein, the phrases “two rounds of target-specifichybridization” or “two rounds of target-specific selection” and theirderivatives refers generally to any process whereby the same targetsequence is subjected to two consecutive rounds of hybridization-basedtarget-specific selection, wherein a target sequence is hybridized to atarget-specific sequence. Each round of hybridization basedtarget-specific selection can include multiple target-specifichybridizations to at least some portion of a target-specific sequence.In one exemplary embodiment, a round of target-specific selectionincludes a first target-specific hybridization involving a first regionof the target sequence and a second target-specific hybridizationinvolving a second region of the target sequence. The first and secondregions can be the same or different. In some embodiments, each round ofhybridization-based target-specific selection can include use of twotarget specific oligonucleotides (e.g., a forward target-specific primerand a reverse target-specific primer), such that each round of selectionincludes two target-specific hybridizations.

As used herein, “comparable maximal minimum melting temperatures” andits derivatives, refers generally to the melting temperature (Tm) ofeach nucleic acid fragment for a single adapter or target-specificprimer after cleavage of the cleavable groups. The hybridizationtemperature of each nucleic acid fragment generated by a single adapteror target-specific primer is compared to determine the maximal minimumtemperature required preventing hybridization of any nucleic acidfragment from the target-specific primer or adapter to the targetsequence. Once the maximal hybridization temperature is known, it ispossible to manipulate the adapter or target-specific primer, forexample by moving the location of the cleavable group along the lengthof the primer, to achieve a comparable maximal minimum meltingtemperature with respect to each nucleic acid fragment.

As used herein, “addition only” and its derivatives, refers generally toa series of steps in which reagents and components are added to a firstor single reaction mixture. Typically, the series of steps excludes theremoval of the reaction mixture from a first vessel to a second vesselin order to complete the series of steps. Generally, an addition onlyprocess excludes the manipulation of the reaction mixture outside thevessel containing the reaction mixture. Typically, an addition-onlyprocess is amenable to automation and high-throughput.

As used herein, “synthesizing” and its derivatives, refers generally toa reaction involving nucleotide polymerization by a polymerase,optionally in a template-dependent fashion. Polymerases synthesize anoligonucleotide via transfer of a nucleoside monophosphate from anucleoside triphosphate (NTP), deoxynucleoside triphosphate (dNTP) ordideoxynucleoside triphosphate (ddNTP) to the 3′ hydroxyl of anextending oligonucleotide chain. For the purposes of this disclosure,synthesizing includes to the serial extension of a hybridized adapter ora target-specific primer via transfer of a nucleoside monophosphate froma deoxynucleoside triphosphate.

As used herein, “polymerizing conditions” and its derivatives, refersgenerally to conditions suitable for nucleotide polymerization. Intypical embodiments, such nucleotide polymerization is catalyzed by apolymerase. In some embodiments, polymerizing conditions includeconditions for primer extension, optionally in a template-dependentmanner, resulting in the generation of a synthesized nucleic acidsequence. In some embodiments, the polymerizing conditions includepolymerase chain reaction (PCR). Typically, the polymerizing conditionsinclude use of a reaction mixture that is sufficient to synthesizenucleic acids and includes a polymerase and nucleotides. Thepolymerizing conditions can include conditions for annealing of atarget-specific primer to a target sequence and extension of the primerin a template dependent manner in the presence of a polymerase. In someembodiments, polymerizing conditions can be practiced usingthermocycling. Additionally, polymerizing conditions can include aplurality of cycles where the steps of annealing, extending, andseparating the two nucleic strands are repeated. Typically, thepolymerizing conditions include a cation such as MgCl₂. Generally,polymerization of one or more nucleotides to form a nucleic acid strandincludes that the nucleotides be linked to each other via phosphodiesterbonds, however, alternative linkages may be possible in the context ofparticular nucleotide analogs.

As used herein, the term “nucleic acid” refers to natural nucleic acids,artificial nucleic acids, analogs thereof, or combinations thereof,including polynucleotides and oligonucleotides. As used herein, theterms “polynucleotide” and “oligonucleotide” are used interchangeablyand mean single-stranded and double-stranded polymers of nucleotidesincluding, but not limited to, 2′-deoxyribonucleotides (nucleic acid)and ribonucleotides (RNA) linked by internucleotide phosphodiester bondlinkages, e.g. 3′-5′ and 2′-5′, inverted linkages, e.g. 3′-3′ and 5′-5′,branched structures, or analog nucleic acids. Polynucleotides haveassociated counter ions, such as H⁺, NH₄ ⁺, trialkylammonium, Mg²⁺, Na⁺and the like. An oligonucleotide can be composed entirely ofdeoxyribonucleotides, entirely of ribonucleotides, or chimeric mixturesthereof. Oligonucleotides can be comprised of nucleobase and sugaranalogs. Polynucleotides typically range in size from a few monomericunits, e.g. 5-40, when they are more commonly frequently referred to inthe art as oligonucleotides, to several thousands of monomericnucleotide units, when they are more commonly referred to in the art aspolynucleotides; for purposes of this disclosure, however, botholigonucleotides and polynucleotides may be of any suitable length.Unless denoted otherwise, whenever a oligonucleotide sequence isrepresented, it will be understood that the nucleotides are in 5′ to 3′order from left to right and that “A” denotes deoxyadenosine, “C”denotes deoxycytidine, “G” denotes deoxyguanosine, “T” denotesthymidine, and “U’ denotes deoxyuridine. Oligonucleotides are said tohave “5′ ends” and “3′ ends” because mononucleotides are typicallyreacted to form oligonucleotides via attachment of the 5′ phosphate orequivalent group of one nucleotide to the 3′ hydroxyl or equivalentgroup of its neighboring nucleotide, optionally via a phosphodiester orother suitable linkage.

As defined herein, the term “nick translation” and its variants comprisethe translocation of one or more nicks or gaps within a nucleic acidstrand to a new position along the nucleic acid strand. In someembodiments, a nick can be formed when a double stranded adapter isligated to a double stranded amplified target sequence. In one example,the primer can include at its 5′ end, a phosphate group that can ligateto the double stranded amplified target sequence, leaving a nick betweenthe adapter and the amplified target sequence in the complementarystrand. In some embodiments, nick translation results in the movement ofthe nick to the 3′ end of the nucleic acid strand. In some embodiments,moving the nick can include performing a nick translation reaction onthe adapter-ligated amplified target sequence. In some embodiments, thenick translation reaction can be a coupled 5′ to 3′ DNApolymerization/degradation reaction, or coupled to a 5′ to 3′ DNApolymerization/strand displacement reaction. In some embodiments, movingthe nick can include performing a DNA strand extension reaction at thenick site. In some embodiments, moving the nick can include performing asingle strand exonuclease reaction on the nick to form a single strandedportion of the adapter-ligated amplified target sequence and performinga DNA strand extension reaction on the single stranded portion of theadapter-ligated amplified target sequence to a new position. In someembodiments, a nick is formed in the nucleic acid strand opposite thesite of ligation.

As used herein, the term “polymerase chain reaction” (“PCR”) refers tothe method of K. B. Mullis U.S. Pat. Nos. 4,683,195 and 4,683,202,hereby incorporated by reference, which describe a method for increasingthe concentration of a segment of a polynucleotide of interest in amixture of genomic DNA without cloning or purification. This process foramplifying the polynucleotide of interest consists of introducing alarge excess of two oligonucleotide primers to the DNA mixturecontaining the desired polynucleotide of interest, followed by a precisesequence of thermal cycling in the presence of a DNA polymerase. The twoprimers are complementary to their respective strands of the doublestranded polynucleotide of interest. To effect amplification, themixture is denatured and the primers then annealed to theircomplementary sequences within the polynucleotide of interest molecule.Following annealing, the primers are extended with a polymerase to forma new pair of complementary strands. The steps of denaturation, primerannealing and polymerase extension can be repeated many times (i.e.,denaturation, annealing and extension constitute one “cycle”; there canbe numerous “cycles”) to obtain a high concentration of an amplifiedsegment of the desired polynucleotide of interest. The length of theamplified segment of the desired polynucleotide of interest (amplicon)is determined by the relative positions of the primers with respect toeach other, and therefore, this length is a controllable parameter. Byvirtue of repeating the process, the method is referred to as the“polymerase chain reaction” (hereinafter “PCR”). Because the desiredamplified segments of the polynucleotide of interest become thepredominant nucleic acid sequences (in terms of concentration) in themixture, they are said to be “PCR amplified”. As defined herein, targetnucleic acid molecules within a sample including a plurality of targetnucleic acid molecules are amplified via PCR. In a modification to themethod discussed above, the target nucleic acid molecules can be PCRamplified using a plurality of different primer pairs, in some cases,one or more primer pairs per target nucleic acid molecule of interest,thereby forming a multiplex PCR reaction. Using multiplex PCR, it ispossible to simultaneously amplify multiple nucleic acid molecules ofinterest from a sample to form amplified target sequences. It is alsopossible to detect the amplified target sequences by several differentmethodologies (e.g., quantitation with a bioanalyzer or qPCR,hybridization with a labeled probe; incorporation of biotinylatedprimers followed by avidin-enzyme conjugate detection; incorporation of³²P-labeled deoxynucleotide triphosphates, such as dCTP or dATP, intothe amplified target sequence). Any oligonucleotide sequence can beamplified with the appropriate set of primers, thereby allowing for theamplification of target nucleic acid molecules from genomic DNA, cDNA,formalin-fixed paraffin-embedded DNA, fine-needle biopsies and variousother sources. In particular, the amplified target sequences created bythe multiplex PCR process as disclosed herein, are themselves efficientsubstrates for subsequent PCR amplification or various downstream assaysor manipulations.

As defined herein “multiplex amplification” refers to selective andnon-random amplification of two or more target sequences within a sampleusing at least one target-specific primer. In some embodiments,multiplex amplification is performed such that some or all of the targetsequences are amplified within a single reaction vessel. The “plexy” or“plex” of a given multiplex amplification refers generally to the numberof different target-specific sequences that are amplified during thatsingle multiplex amplification. In some embodiments, the plexy can beabout 12-plex, 24-plex, 48-plex, 96-plex, 192-plex, 384-plex, 768-plex,1536-plex, 3072-plex, 6144-plex or higher.

In some embodiments, the disclosure relates generally to methods,compositions, systems, apparatuses and kits for avoiding or reducing theformation of amplification artifacts (for example primer-dimers) duringselective amplification of one or more target nucleic acid molecules ina population of nucleic acid molecules.

In some embodiments, the disclosure relates generally to theamplification of multiple target-specific sequences from a population ofnucleic acid molecules. In some embodiments, the method compriseshybridizing one or more target-specific primer pairs to the targetsequence, extending a first primer of the primer pair, denaturing theextended first primer product from the population of nucleic acidmolecules, hybridizing to the extended first primer product the secondprimer of the primer pair, extending the second primer to form a doublestranded product, and digesting the target-specific primer pair awayfrom the double stranded product to generate a plurality of amplifiedtarget sequences. In some embodiments, the digesting includes partialdigesting of one or more of the target-specific primers from theamplified target sequence. In some embodiments, the amplified targetsequences can be ligated to one or more adapters. In some embodiments,the adapters can include one or more DNA barcodes or tagging sequences.In some embodiments, the amplified target sequences once ligated to anadapter can undergo a nick translation reaction and/or furtheramplification to generate a library of adapter-ligated amplified targetsequences.

In some embodiments, the disclosure relates generally to the preparationand formation of multiple target-specific amplicons. In someembodiments, the method comprises hybridizing one or moretarget-specific primer pairs to a nucleic acid molecule, extending afirst primer of the primer, pair, denaturing the extended first primerfrom the nucleic acid molecule, hybridizing to the extended first primerproduct, a second primer of the primer pair and extending the secondprimer, digesting the target-specific primer pairs to generate aplurality of target-specific amplicons. In some embodiments, adapterscan be ligated to the ends of the target-specific amplicons prior toperforming a nick translation reaction to generate a plurality oftarget-specific amplicons suitable for nucleic acid sequencing. In someembodiments, the one or more target specific amplicons can be amplifiedusing bridge amplification or emPCR to generate a plurality of clonaltemplates suitable for nucleic acid sequencing. In some embodiments, thedisclosure generally relates to methods for preparing a target-specificamplicon library, for use in a variety of downstream processes or assayssuch as nucleic acid sequencing or clonal amplification. In oneembodiment, the disclosure relates to a method of performingtarget-specific multiplex PCR on a nucleic acid sample having aplurality of target sequences using primers having a cleavable group.

In one embodiment, nucleic acid templates to be sequenced using the IonTorrent PGM™ or Ion Torrent Proton™ system can be prepared from apopulation of nucleic acid molecules using the target-specificamplification techniques as outlined herein. Optionally, followingtarget-specific amplification a secondary and/or tertiary amplificationprocess including, but not limited to, a library amplification stepand/or a clonal amplification step such as emPCR can be performed.

In some embodiments, the disclosure relates to a composition comprisinga plurality of target-specific primer pairs, each containing a forwardprimer and a reverse primer having at least one cleavable group locatedat either a) the 3′ end or the 5′ end, and/or b) at about the centralnucleotide position of the target-specific primer, and wherein thetarget-specific primer pairs can be substantially non-complementary toother primer pairs in the composition. In some embodiments, thecomposition comprises at least 1000, 2000, 3000, 4000, 6000, 9000,12000, or more target-specific primer pairs. In some embodiments, thetarget-specific primer pairs comprise about 15 nucleotides to about 40nucleotides in length, wherein at least one nucleotide is replaced witha cleavable group. In some embodiments the cleavable group can be auridine nucleotide. In some embodiments, the target-specific primer setsare designed to amplify an exon, gene, exome or region of the genomeassociated with a clinical or pathological condition, e.g., theamplification of one or more single nucleotide mutations (SNPs)associated with cancer, such as colon cancer, or the amplification ofmutations associated with an inherited disease such as cystic fibrosis.In some embodiments, the target-specific primer pairs when hybridized toa target sequence and amplified as outlined herein can generate alibrary of adapter-ligated amplified target sequences that are about 100to about 500 base pairs in length. In some embodiments, no oneadapter-ligated amplified target sequence is overexpressed in thelibrary by more than 30% as compared to the remainder of theadapter-ligated amplified target sequences in the library. In someembodiments, the adapter-ligated amplified target sequence library issubstantially homogenous with respect to GC content, amplified targetsequence length or melting temperature (Tm).

In some embodiments, the disclosure relates generally to a kit forperforming multiplex PCR comprising a plurality of target-specificprimers having a cleavable group, a DNA polymerase, an adapter, dATP,dCTP, dGTP and dTTP. In some embodiments, the cleavable group can be auracil nucleotide. The kit can further include one or more antibodies,nucleic acid barcodes, purification solutions or columns.

In some embodiments, the disclosure relates to a kit for generating atarget-specific amplicon library comprising a plurality oftarget-specific primers having a cleavable group, a DNA polymerase, anadapter, dATP, dCTP, dGTP, dTTP, and a cleaving reagent. In someembodiments, the kit further comprises one or more antibodies, nucleicacid barcodes, purification solutions or columns.

In one embodiment, the disclosure generally relates to the amplificationof multiple target-specific sequences from a single nucleic acid sourceor sample. In another embodiment, the disclosure relates generally tothe target-specific amplification of two or more target sequences fromtwo or more nucleic acid sources, samples or species. For example, it isenvisioned by the disclosure that a single nucleic acid sample caninclude genomic DNA or fixed-formalin paraffin-embedded (FFPE) DNA. Itis also envisioned that the sample can be from a single individual, acollection of nucleic acid samples from genetically related members,multiple nucleic acid samples from genetically unrelated members,multiple nucleic acid samples (matched) from a single individual such asa tumor sample and normal tissue sample, or genetic material from asingle source that contains two distinct forms of genetic material suchas maternal and fetal DNA obtained from a maternal subject, or thepresence of contaminating bacteria DNA in a sample that contains plantor animal DNA. In some embodiments, the source of nucleic acid materialcan include nucleic acids obtained from a newborn, for example astypically procured as a blood sample for newborn screening.

The nucleic acid sample can include high molecular weight material suchas genomic DNA or cDNA. The sample can include low molecular weightmaterial such as nucleic acid molecules obtained from FFPE or archivedDNA samples. In another embodiment, low molecular weight materialincludes enzymatically or mechanically sheared DNA. The sample caninclude cell-free circulating DNA such as material obtained from amaternal subject. In some embodiments, the sample can include nucleicacid molecules obtained from biopsies, tumors, scrapings, swabs, blood,mucus, urine, plasma, semen, hair, laser capture micro-dissections,surgical resections, and other clinical or laboratory obtained samples.In some embodiments, the sample can be an epidemiological, agricultural,forensic or pathogenic sample.

In some embodiments, the sample can include nucleic acid moleculesobtained from an animal such as a human or mammalian source. In anotherembodiment, the sample can include nucleic acid molecules obtained froma non-mammalian source such as a plant, bacteria, virus or fungus. Insome embodiments, the source of the nucleic acid molecules may be anarchived or extinct sample or species.

In some embodiments, the disclosure relates generally to the selectiveamplification of at least one target sequence in a normal or diseasedcontaining tissue, biopsy, core, tumor or other sample. In someembodiments, the disclosure generally relates to the selectiveamplification of at least one target sequence and the detection and/oridentification of mutations in the diseased tissue, core, biopsy ortumor sample. In some embodiments, the diseased or normal sample caninclude whole genomic DNA, formalin-fixed paraffin-embedded tissue(FFPE), sheared or enzymatically treated DNA. In some embodiments, thedisclosure is directed to the selective amplification of at least onetarget sequence and detection and/or identification of clinicallyactionable mutations. In some embodiments, the disclosure is directed tothe detection and/or identification of mutations associated with drugresistance or drug susceptibility. In some embodiments, the disclosureis generally directed to the identification and/or quantitation ofgenetic markers associated with organ transplantation or organrejection.

In some embodiments, the disclosure relates generally to the selectiveamplification of at least one target sequence in cell-free circulatingDNA. In some embodiments, the selective amplification of at least onetarget sequence in a sample includes a mixture of different nucleic acidmolecules. The selective amplification can optionally be accompanied bydetection and/or identification of mutations observed in circulatingDNA. In some embodiments, the selective amplification can optionally beaccompanied by detection and/or identification of mutations associatedwith cancer or an inherited disease such as metabolic, neuromuscular,developmental, cardiovascular, autoimmune or other inherited disorder.

In some embodiments, the target-specific primers and primer pairs aretarget-specific sequences that can amplify specific regions of a nucleicacid molecule. In some embodiments, the target-specific primers canamplify genomic DNA or cDNA. In some embodiments, the target-specificprimers can amplify mammalian DNA, such as human DNA. In someembodiments, the amount of DNA required for selective amplification canbe from about 1 ng to 1 microgram. In some embodiments, the amount ofDNA required for selective amplification of one or more target sequencescan be about 1 ng, about 5 ng or about 10 ng. In some embodiments, theamount of DNA required for selective amplification of target sequence isabout 10 ng to about 200 ng.

In some embodiments, selective amplification of at least one targetsequence further includes nucleic acid sequencing of the amplifiedtarget sequence. Optionally, the method further includes detectingand/or identifying mutations present in the sample identified throughnucleic acid sequencing of the amplified target sequence.

In some embodiments, target sequences or amplified target sequences aredirected to mutations associated with cancer. In some embodiments, thetarget sequences or amplified target sequences are directed to mutationsassociated with one or more cancers selected from the group consistingof head and neck cancers, brain cancer, breast cancer, ovarian cancer,cervical cancer, colorectal cancer, endometrial cancer, gallbladdercancer, gastric cancer, bladder cancer, prostate cancer, testicularcancer, liver cancer, lung cancer, kidney (renal cell) cancer,esophageal cancer, pancreatic cancer, thyroid cancer, bile duct cancer,pituitary tumor, wilms tumor, kaposi sarcoma, osteosarcoma, thymuscancer, skin cancer, heart cancer, oral and larynx cancer, leukemia,neuroblastoma and non-hodgkin lymphoma. In one embodiment, the mutationscan include substitutions, insertions, inversions, point mutations,deletions, mismatches and translocations. In one embodiment, themutations can include variation in copy number. In one embodiment, themutations can include germline or somatic mutations. In one embodiment,the mutations associated with cancer are located in at least one of thegenes provided in Tables 1 or 4, or provided in Table 7 of U.S.Application No. 61/598,881 hereby incorporated by reference in itsentirety. In some embodiments, the mutations can be any of the genomiccoordinates provided in Table 18, or provided in Table 7 of U.S.Application 61/598,881 hereby incorporated by reference in its entirety.In some embodiments, the target sequences directed to mutationsassociated with cancer can include any one or more of the mutationsprovided in Table 10. In some embodiments, the mutations can be foundwithin any one or more of the genomic coordinates provided in Table 16or Table 18.

In some embodiments, the mutations associated with cancer are located inat least one of the genes selected from ABI1; ABL1; ABL2; ACSL3; ACSL6;AFF1; AFF3; AFF4; AKAP9; AKT1; AKT2; ALK; APC; ARHGAP26; ARHGEF12;ARID1A; ARNT; ASPSCR1; ASXL1; ATF1; ATIC; ATM; AXIN2; BAP1; BARD1;BCAR3; BCL10; BCL11A; BCL11B; BCL2; BCL3; BCL6; BCL7A; BCL9; BCR; BIRC3;BLM; BMPR1A; BRAF; BRCA1; BRCA2; BRD3; BRD4; BRIP1; BUB1B; CARD11; CARS;CASC5; CBFA2T3; CBFB; CBL; CBLB; CBLC; CCDC6; CCNB1IP1; CCND1; CCND2;CD74; CD79A; CDC73; CDH1; CDH11; CDK4; CDK6; CDKN2A; CDKN2B; CDKN2C;CDX2; CEBPA; CEP110; CHEK1; CHEK2; CHIC2; CHN1; CIC; CITTA; CLP1; CLTC;CLTCL1; COL1A1; CREB1; CREB3L2; CREBBP; CRTC1; CRTC3; CSF1R; CTNNB1;CXCR7; CYLD; CYTSB; DCLK3; DDB2; DDIT3; DDR2; DDX10; DDX5; DDX6; DEK;DGKG; DICER1; DNMT3A; EGFR; EIF4A2; ELF4; ELL; ELN; EML4; EP300; EPS15;ERBB2; ERBB4; ERC1; ERCC2; ERCC3; ERCC4; ERCC5; ERG; ETV1; ETV4; ETV5;ETV6; EWSR1; EXT1; EXT2; EZH2; FAM123B; FANCA; FANCC; FANCD2; FANCE;FANCF; FANCG; FAS; FBXW7; FCRL4; FGFR1; FGFR10P; FGFR2; FGFR3; FH;FIP1L1; FLCN; FLI1; FLT1; FLT3; FNBP1; FOXL2; FOXO1; FOXO3; FOXO4;FOXP1; FUS; GAS7; GATA1; GATA2; GATA3; GMPS; GNAQ; GNAS; GOLGA5; GOPC;GPC3; GPHNGPR124; HIP1; HIST1H4I; HLF; HNF1A; HNRNPA2B1; HOOK3; HOXA11;HOXA13; HOXA9; HOXC11; HOXC13; HOXD13; HRAS; HSP90AA1; HSP90AB1; IDH1;IDH2; IKZF1; IL2; IL21R; IL6ST; IRF4; ITGA10; ITGA9; ITK; JAK1; JAK2;JAK3; KDM5A; KDM5C; KDM6A; KDR; KDSR; KIAA1549; KIT; KLF6; KLK2; KRAS;KTN1; LASP1; LCK; LCP1; LHFP; LIFR; LMO2; LPP; MAF; MALT1; MAML2;MAP2K1; MAP2K4; MDM2; MDM4; MECOM; MEN1; MET; MITF; MKL1; MLH1; MLL;MLLT1; MLLT10; MLLT3; MLLT4; MLLT6; MN1; MPL; MRE11A; MSH2; MSH6; MSI2;MSN; MTCP1; MTOR; MUC1; MYB; MYC; MYCL1; MYCN; MYH11; MYH9; MYST3;MYST4; NACA; NBN; NCOA1; NCOA2; NCOA4; NEK9; NF1; NF2; NFE2L2; NFKB2;NIN; NKX2-1; NLRP1; NONO; NOTCH1; NOTCH2; NPM1; NR4A3; NRAS; NSD1;NTRK1; NTRK3; NUMA1; NUP214; NUP98; OLIG2; OMD; PAFAH1B2; PALB2; PATZ1;PAX3; PAX5; PAX7; PAX8; PBRM1; PBX1; PCM1; PDE4DIP; PDGFB; PDGFRA;PDGFRB; PER1; PHOX2B; PICALM; PIK3CA; PIK3R1; PIM1; PLAG1; PML; PMS1;PMS2; POU2AF1; POU5F1; PPARG; PPP2R1A; PRCC; PRDM16; PRF1; PRKAR1A;PRRX1; PSIP1; PTCH1; PTEN; PTPN11; RABEP1; RAD50; RAD51L1; RAF1;RANBP17; RAP1GDS1; RARA; RBI; RBM15; RECQL4; REL; RET; RHOH; RNF213;ROS1; RPN1; RPS6KA2; RUNX1; RUNX1T1; SBDS; SDHAF2; SDHB; SETD2; SFPQ;SFRS3; SH3GL1; SLC45A3; SMAD4; SMARCA4; SMARCB1; SMO; SOCS1; SRC;SRGAP3; SS18; SS18L1; STIL; STK11; STK36; SUFU; SYK; TAF15; TAF1L; TAL1;TAL2; TCF12; TCF3; TCL1A; TET1; TET2; TEX14; TFE3; TFEB; TFG; TFRC;THRAP3; TLX1; TLX3; TMPRSS2; TNFAIP3; TOP1; TP53; TPM3; TPM4; TPR;TRIM27; TRIM33; TRIP11; TSC1; TSC2; TSHR; USP6; VHL; WAS; WHSC1L1; WRN;WT1; XPA; XPC; ZBTB16; ZMYM2; ZNF331; ZNF384; and ZNF521.

In some embodiments, the mutations associated with cancer are located inat least one of the genes selected from ABL1; AKT1; ALK; APC; ATM; BRAF;CDH1; CDKN2A; CSF1R; CTNNB1; EGFR; ERBB2; ERBB4; FBXW7; FGFR1; FGFR2;FGFR3; FLT3; GNAS; HNF1A; HRAS; IDH1; JAK2; JAK3; KDR; KIT; KRAS; MET;MLH1; MPL; NOTCH1; NPM1; NRAS; PDGFRA; PIK3CA; PTEN; PTPN11; RB1; RET;SMAD4; SMARCB1; SMO; SRC; STK11; TP53; and VHL.

In some embodiments, the amplified target sequences are directed to anyone of more of the genomic coordinates provided in Table 18. In someembodiments, any one or more of the cancer target-specific primersprovided in Tables 2, 3 or 17 can be used to amplify a target sequencepresent in a sample as disclosed by the methods described herein.

In some embodiments, the cancer target-specific primers from Tables 2, 3or 17 can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 40, 60, 80, 100, 150,200, 400, 500, 800, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000,9000, 10,000, 11,000, 12,000, 13,000 or more, target-specific primers.In some embodiments, the amplified target sequences can include any oneor more of the amplified target sequences generated at the genomiccoordinates (using amplicon ID target-specific primers) provided inTables 10 or 18. In some embodiments, at least one of thetarget-specific primers associated with cancer is at least 90% identicalto at least one nucleic acid sequence selected from SEQ ID NOs:1-103,143. In some embodiments, at least one of the target-specificprimers associated with cancer is complementary across its entire lengthto at least one target sequence in a sample. In some embodiments, atleast one of the target-specific primers associated with cancer includesa non-cleavable nucleotide at the 3′ end. In some embodiments, thenon-cleavable nucleotide at the 3′ end includes the terminal 3′nucleotide. In one embodiment, the amplified target sequences aredirected to individual exons having a mutation associated with cancer.In some embodiments, the disclosure relates generally to the selectiveamplification of more than one target sequences in a sample and thedetection and/or identification of mutations associated with cancer. Insome embodiments, the amplified target sequences include two or morenucleotide sequences provided in Table 2. In some embodiments, theamplified target sequences can include any one or more the amplifiedtarget sequences generated at the genomic coordinates using the ampliconID target-specific primers provided in Table 18, or provided in Table 7of U.S. Application 61/598,881 hereby incorporated by reference in itsentirety. In one embodiment, the amplified target sequences include 100,200, 500, 1000, 2000, 3000, 6000, 8000, 10,000, 12,000, or moreamplicons from Tables 1-5, or Tables 6 and 7 of U.S. Application61/598,881 hereby incorporated by reference in their entireties. In someembodiments, the disclosure relates generally to the detection andoptionally, the identification of clinically actionable mutations. Asdefined herein, the term “clinically actionable mutations” includesmutations that are known or can be associated by one of ordinary skillin the art with, but not limited to, prognosis for the treatment ofcancer. In one embodiment, prognosis for the treatment of cancerincludes the identification of mutations associated with responsivenessor non-responsiveness of a cancer to a drug, drug combination, ortreatment regime. In one embodiment, the disclosure relates generally tothe amplification of a plurality of target sequences from a populationof nucleic acid molecules linked to, or correlated with, the onset,progression or remission of cancer.

In some embodiments, target-specific primers are designed using theprimer criteria disclosed herein. In some embodiments, target-specificprimers are designed using the primer criteria disclosed herein anddirected to one or more genes associated with breast cancer. In someembodiments, target-specific primers associated with breast cancerinclude at least one target-specific primer selected from one or moregenes selected from the group consisting of AIM1, AR, ATM, BARD1, BCAS1,BRIP1, CCND1, CCND2, CCNE1, CDH1, CDK3, CDK4, CDKN2A, CDKN2B, CAMK1D,CHEK2, DIRAS3, EGFR, ERBB2, EPHA3, ERBB4, ETV6, GNRH1, KCTD9, CDCA2,EBF2, EMSY, BNIP3L, PNMA2, DPYSL2, ADRA1A, STMN4, TRIM35, PAK1, AQP11,CLSN1A, RSF1, KCTD14, THRSP, NDUFC2, ALG8, KCTD21, USP35, GAB2, DNAH9,ZNF18, MYOCD, STK11, TP53, JAK1, JAK2, MET, PDGFRA, PML, PTEN, RET,TMPRSS2, WNK1, FGFR1, IGF1R, PPP1R12B, PTPRT, GSTM1, IPO8, MYC, ZNF703,MDM1, MDM2, MDM4, MKK4, P14 KB, NCOR1, NBN, PALB2, RAD50, RAD51, PAK1,RSF1, INTS4, ZMIZ1, SEPHS1, FOXM1, SDCCAG1, IGF1R, TSHZ2, RPSK6K1,PPP2R2A, MTAP, MAP2K4, AURKB, BCL2, BUB1, CDCA3, CDCA4, CDC20, CDC45,CHEK1, FOXM1, HDAC2, IGF1R, KIF2C, KIFC1, KRAS, RB1, SMAD4, NCOR1, UTX,MTHDFD1L, RAD51AP1, TTK and UBE2C.

In some embodiments, the disclosure relates generally to theamplification of target sequences directed to mutations associated witha congenital or inherited disease. In some embodiments, the disclosurecan include the amplification of target sequences directed to somatic orgermline mutations. In some embodiments, the mutations can be autosomaldominant or autosomal recessive. In one embodiment, the mutationsassociated with a congenital or inherited disease are located in atleast one of the genes or diseases provided in Table 4. In someembodiments, the disclosure relates to the amplification of targetsequences in a sample associated with one or more inherited diseasesselected from the group consisting of Adenosine AminohydrolaseDeficiency (ADA); Agammaglobulinemia, X-linked, Type 1; AlagilleSyndrome; All Hypertrophic and Dilated Cardiomyopathy; AlopeciaUniversalis Congenita (ALUNC); Alpers Syndrome; Alpha-1-AntitrypsinDeficiency; Alpha-Thalassemia—Southeast Asia; Amyotrophic LateralSclerosis—Lou Gehrig's Disease; Androgen Insensitivity Syndrome;Aniridia; Ankylosing spondylitis; APC-Associated Polyposis Conditions;Argininosuccinate Lyase Deficiency; Arrhythmogenic Right VentricularDysplasia/Cardiomyopathy; Ataxia with Oculomotor Apraxia Type 2; Ataxiawith Vitamin E Deficiency; Ataxia-Telangiectasia; AutoimmunePolyendocrine Syndrome; Beta-Hydroxyisobutyryl CoA Deacylase deficiency(HIBCH deficiency); Biotimidase Deficiency;Blepharophimosis-ptosis-epicanthus inversus; Bloom Syndrome;Brachydactyly; Brachydactyly—Hypertension Syndrome; Brachydactyly TypeB1; Branchiootorenal Spectrum Disorders; BRCA1; Campomelic Dysplasia;Canavan; Cerebrotendinous Xanthomatosis; Ceroid-lipofuscinoses-Batton;Charcot-Marie-Tooth Disease Type 2B; Charcot-Marie-Tooth Neuropathy Type1B; Charcot-Marie-Tooth Neuropathy Type 2A2; Charge Syndrome; Cherubism;Choroideremia; Citrin Deficiency; Citrullinemia Type I; Coffin-LowrySyndrome; Cohen Syndrome; Collagen 4A5; Common Variable ImmuneDeficiency; Congenital Adrenal Hyperplasia; Congenital Cataracts, FacialDysmorphism, and Neuropathy; Congenital Disorder of Glycosylation Type1a; Congenital Myasthenic Syndromes; Cornelia de Lange Syndrome; Cysticfibrosis; Cystinosis; Darier Disease; Desmin Storage Myopathy; DFNA2Nonsyndromic Hearing Loss; Diamond-Blackfan Anemia; Double CortexSyndrome; Duane Syndrome; Duchenne/Becker muscular dystrophy;Dysferlinopathy; Dyskeratosis Congenita; Early-Onset Familial AlzheimerDisease; Early-Onset Primary Dystonia (DYT1); Ehlers Danlos;Ehlers-Danlos Syndrome, Classic Type; Ehlers-Danlos Syndrome,Hypermobility Type; Ehlers-Danlos Syndrome, Kyphoscoliotic Form;Emery-Dreifuss Muscular Dystrophy X linked; Epidermolysis BullosaSimplex; Fabry Disease; Facioscapulohumeral Muscular Dystrophy; FamilialDysautonomia (HSAN III); Familial Hyperinsulinism (FHI); FamilialHypertrophic Cardiomyopathy; Familial Transthyretin Amyloidosis; FanconiAnemia; Fragile X; Friedreich Ataxia; FRMD7-Related Infantile Nystagmus;Fryns Syndrome; Galactosemia; Gaucher Disease; Glycine Encephalopathy;Glycogen Storage Disease Type VI; Hemophagocytic Lymphohistiocytosis;Hemophilia A; Hemophilia B; Hepatic Veno-Occlusive Disease withImmunodeficiency; Hereditary Hemorrhagic Telangiectasia; HereditaryNeuropathy with Liability to Pressure Palsies; Hereditary NonpolyposisColon Cancer; Hexosaminidase A Deficiency; HFE-Associated HereditaryHemochromatosis; Holt-Oram Syndrome; Huntington Disease;Hydroxymethylbilane Synthase (HMBS) Deficiency; Hypophosphatasia;Inclusion Body Myopathy 2; Incontinentia Pigmenti; Juvenile PolyposisSyndrome; Kallmann Syndrome; Leber Congenital Amaurosis; Lebercongenital amaurosis 10; L1-Fraumeni Syndrome; Limb-Girdle MuscularDystrophy Type 2A Calpainopathy; LIS1-Associated Lissencephaly; Long QTSyndrome; Lowe Syndrome; Malignant Hyperthermia Susceptibility; MapleSyrup Urine Disease; MAPT-Related Disorders; McKusick-Kaufman Syndrome;MECP2-Rett Syndrome; Menkes; Metachromatic Leukodystrophy; MethylmalonicAcidemia; Mucolipidosis II; Multiple Endocrine Neoplasia Type 1;Multiple Endocrine Neoplasia Type 2; Myotonia Congenita; MyotonicDystrophy Type 1; Myotonic Dystrophy Type 2; Nail-Patella Syndrome;Nemaline Myopathy; Neurofibromatosis 1; Neurofibromatosis 2; NoonanSyndrome; Ocular Albinism, X-Linked; Oculocutaneous Albinism Type 1;Oculocutaneous Albinism Type 2; Oculopharyngeal Muscular Dystrophy;Optic Atrophy Type 1; Ornithine Transcarbamylase Deficiency;Osteogenesis Imperfecta; Parkinson Disease; Pendred Syndrome; PeroxisomeBiogenesis, Zellweger; Phenylketonuria; Polycystic Kidney Disease; PompeDisease-GSD II; Primary Ciliary Dyskinesia; Retinitis Pigmentosa;Retinoblastoma; Saethre-Chotzen Syndrome; SCN9A-Related InheritedErythromelalgia; SHOX-Related Haploinsufficiency; Sickle Cell Disease;Smith-Lemli-Opitz Syndrome; Smith-Magenis Syndrome; Sotos Syndrome;Spastic Paraplegia 3A; Spastic Paraplegia 7; Spastic Paraplegia 8;Spastic Paraplegia Type 1; Spastic Paraplegia Type 4; Spinal MuscularAtrophy; Spinocerebellar Ataxia 2; Spinocerebellar Ataxia 3;Spinocerebellar Ataxia 7; Spinocerebellar Ataxia Type 1; SticklerSyndrome; Thanatophoric Dysplasia; Thoracic Aortic Aneurysms and AorticDissections; Treacher Collins Syndrome; Trimethylaminuria; TuberousSclerosis Complex; Udd Distal Myopathy; Usher Syndrome type 1; Very LongChain Acyl-Coenzyme A Dehydrogenase Deficiency; von Hippel-Lindau;Waardenburg Syndrome, Type 1; Werner Syndrome; Wilms Tumor; WilsonDisease; Wiskott-Aldrich; X-Linked Adrenal Hypoplasia Congenita;X-Linked Adrenoleukodystrophy; X-Linked Dystonia-Parkinsonism; X-linkedJuvenile Retinoschisis; X-linked myotubular Myopathy; X-Linked SCIDS;and Zellweger Syndrome.

In one embodiment, the mutations associated with a congenital orinherited disease can include substitutions, insertions, inversions,point mutations, deletions, mismatches and translocations. In someembodiments, the mutations associated with an inherited or congenitaldisease includes copy number variation. In some embodiments, thedisclosure relates generally to the selective amplification of at leastone target sequence and the detection and/or identification of mutationsassociated with an inherited disease. In some embodiments, the mutationsassociated with a congenital or inherited disease can be located in oneor more of the genes selected from the group consisting of ABCA4; ABCC8;ABCD1; ACADVL; ACTA2; ACTC; ACTC1; ACVRL1; ADA; AIPL1; AIRE; ALK1; ALPL;AMT; APC; APP; APTX; AR; ARL6; ARSA; ASL; ASPA; ASS; ASS1; ATL; ATM;ATP2A2; ATP7A; ATP7B; ATXN1; ATXN2; ATXN3; ATXN7; BBS6; BCKDHA; BCKDHB;BEST1; BMPR1A; BRCA1; BRCA2; BRIP1; BTD; BTK; C2orf25; CA4; CALR3;CAPN3; CAV3; CCDC39; CCDC40; CDH23; CEP290; CERKL; CFTR; CHAT; CHD7;CHEK2; CHM; CHRNA1; CHRNB1; CHRND; CHRNE; CLCN1; CNBP; CNGB1; COH1;COL11A1; COL11A2; COL1A1; COL1A2; COL2A1; COL3A1; COL4A5; COL5A1;COL5A2; COL7A1; COL9A1; CRB1; CRX; CTDP1; CTNS; CYP21A2; CYP27A1; DAX1;DBT; DCX; DES; DHCR7; DJ1; DKC1; DLD; DMD; DMPK; DNAAF1; DNAAF2; DNAH11;DNAH5; DNA11; DNAI2; DNAL1; DNM12; DOK7; DSC2; DSG2; DSP; DYSF; DYT1;EMD; ENG; EYA1; EYS; F8; F9; FANCA; FANCC; FANCF; FANCG; FANCJ; FANDC2;FBN1; FBXO7; FGFR1; FGFR3; FMO3; FMR1; FOXL2; FRG1; FRMD7; FSCN2; FXN;GAA; GALT; GBA; GBE1; GCSH; GDF5; GJB2; GJB3; GJB6; GLA; GLDC; GNE;GNPTAB; GPC3; GPR143; GUCY2D; HBA1; HBA2; HBB; HD; HERG; HEXA; HFE; HHF;HIBCH; HLA-B27; HMBS; HPLH1; HPRP3; HR; HTNB; HTT; IKBKAP; IKBKG; IL2RG;IMPDH1; ITGB4; JAG1; JPH3; KCNE1; KCNE2; KCNH2; KCNQ1; KCNQ4; KIAA0196;KLHL7; KRAS; KRT14; KRT5; L1CAM; LAMB3; LAMP2; LDB3; LMNA; LMX18; LRAT;LRRK2; MAPT; MC1R; MECP2; MED12; MEN1; MERTK; MFN2; MKKS; MLH1; MMAA;MMAB; MMACHC; MMADHC; MPZ; MSH2; MTM1; MTND5; MTTG; MTTI; MTTK; MTTL1;MTTQ; MUT; MYBPC3; MYH11; MYH6; MYH7; MYL2; MYL3; MYLK2; MYO7A; ND5;ND6; NEMO; NF1; NF2; NIPBL; NR0B1; NR2E3; NRAS; NSD1; OCA2; OCRL; OPA1;OTC; PABPN1; PAFAH1B1; PAH; PARK2; PARK7; PARKIN; PAX3; PAX6; PCDH15;PEX1; PEX2; PEX10; PEX13; PEX14; PEX19; PEX26; PEX3; PEX5; PINK1; PKD1;PKD2; PKD3; PKHD1; PKP2; PLEC1; PLOD1; PMM2; PMP22; POLG; PPT1; PRCD;PRKAG2; PRNP; PROM1; PRPF3; PRPF8; PRPH2; PRPN; PSEN1; PSEN2; PTCH1;PTPN11; RAB7A; RAF1; RAI1; RAPSN; RB1; RDH12; RDS; RECQL3; RET; RHO;ROR2; RP1; RP2; RP9; RPE65; RPGR; RPGRIP1; RPL11; RPL35A; RPS10; RPS17;RPS19; RPS24; RPS26; RPS6KA3; RPS7; RPSL5; RS1; RSPH4A; RSPH9; RYR1;RYR2; SALL4; SCA3; SCN5A; SCN9A; SEMA4A; SERPINA1; SERPING1; SGCD;SH3BP2; SHOX; SIX1; SIX5; SLC25A13; SLC25A4; SLC26A4; SMAD4; SMN1; SNCA;SNRNP200; SOD1; SOS1; SOX9; SP110; SPAST; SPATA7; SPG3A; SPG4; SPG7;TAF1; TBX5; TCOF1; TGFBR1; TGFBR2; TNFRSC13C; TNNC1; TNNI3; TNNT1;TNNT2; TNXB; TOPORS; TOR1A; TP53; TPM1; TRNG; TRNI; TRNK; TRNL1; TRNQ;TSC1; TSC2; TTN; TTPA; TTR; TULP1; TWIST1; TXNDC3; TYR; USH1C; USH1H;USH2A; VCL; VHL; VPS13B; WAS; WRN; WT1; and ZNF9.

In some embodiments, target-specific primers directed to one or moreinherited diseases or congenital disorders can be selected from any oneor more of the target-specific primers provided in Table 15. In someembodiments, the target-specific primers from Table 15 can include 2, 3,4, 5, 6, 7, 8, 9, 10, 20, 40, 60, 80, 100, 150, 200, 400, 500, 800,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 11,000,12,000, 13,000 or more target-specific primers. In some embodiments, theamplified target sequences can include any one or more of the amplifiedtarget sequences generated at the genomic coordinates (using amplicon IDtarget-specific primers) provided in Table 16. In some embodiments, atleast one of the target-specific primers associated with a congenitaldisease or disorder is at least 90% identical to at least one nucleicacid sequence selected from SEQ ID NOs: 1-103,143. In some embodiments,at least one of the target-specific primers associated with a congenitaldisease or disorder is complementary across its entire length to atleast one target sequence in a sample. In some embodiments, at least oneof the target-specific primers associated with a congenital disease ordisorder includes a non-cleavable nucleotide at the 3′ end. In someembodiments, the non-cleavable nucleotide at the 3′ end includes theterminal 3′ nucleotide. In one embodiment, target sequences or resultingamplified target sequences are directed to individual exons having amutation associated with an inherited disease. In some embodiments,congenital or inherited disease amplified target sequences can includetwo or more target-specific primers provided in Table 15 (or table 8 ofU.S. Application 61/598,881 hereby incorporated by reference in itsentirety). In some embodiments, the amplified target sequences caninclude any one or more of the amplified target sequences generated atthe genomic coordinates using the amplicon ID target-specific primersprovided in Table 16 (or table 9 of U.S. Application 61/598,881 herebyincorporated by reference in its entirety). In some embodiments,congenital or inherited disease amplified target sequences can includetwo or more target-specific primers provided in Table 15. In someembodiments, the amplified target sequences can include any one or moreof the amplified target sequences generated at the genomic coordinatesusing the amplicon ID target-specific primers provided in Table 16. Inone embodiment, the congenital or inherited disease target-specificprimers can include any one or more of the target-specific primersprovided in Table 8 of U.S. Application 61/598,881 hereby incorporatedby reference in its entirety. In some embodiments, any one or moretarget-specific primers can be designed using the target-specific primerselection criteria outlined herein. In some embodiments, the disclosurerelates generally to the selective amplification of more than one targetsequence in a sample and the detection and/or identification ofmutations associated with a congenital or inherited disease. In oneembodiment, the disclosure relates generally to the amplification of aplurality of target sequences linked to, or correlated with a congenitalor inherited disease.

In some embodiments, the target-specific primers are prepared to amplifyregions or fragments of the human genome associated with a congenital orinherited disease. In some embodiments, the target-specific primers canbe prepared to amplify regions of the human genome associated withheredity disorders, such as cystic fibrosis, Alagille syndrome, Alperssyndrome, Alpha-Thalassemia, Amyotrophic Lateral Sclerosis, Anklosingspondylitis, Ataxia-Telangiectasia, congenital Myasthenic syndromes,Darier disease, Diamond-Blackfan anemia, early onset familial Alzheimerdisease, Ehlers-Danlos syndrome, Epidermolysis Bullosa Simplex, familialHypertrophic Cardiomyopathy, Fanconi anemia, Glycine Encephalopathy,Hereditary Hemorrhagic Telangiectasia, Huntington Disease, JuvenilePolyposis syndrome, Leber Congenital Amaurosis, Long QT syndrome, MapleSyrup Urine Disease, Marfan syndrome, Mitochondrial Encephalomyopathy,Methylmalonic Acidemia, Multiple Endocrine Neoplasia Type 2, Noonansyndrome, Parkinson disease, Peroxisome Biogenesis, Primary CilaryDyskineasia, Retinitis Pigmentosa, Stickler syndrome, Thoracic AorticAneurysms and Aortic Dissections, Tuberous Sclerosis Complex, Ushersyndrome, Werner syndrome, Wilson disease and Zellweger syndrome. Insome embodiments, the target-specific primers can be prepared from anyone or more of the genes provided in Table 4.

In some embodiments, the disclosure relates generally to detecting thepresence of a target sequence or amplified target sequence associatedwith one or more newborn disorders. In some embodiments, the disclosurerelates generally to detecting the presence of an amplified targetsequence obtained by amplifying a sample containing at least one targetsequence associated with a newborn disorder with one or moretarget-specific primers disclosed herein. In some embodiments, thedisclosure relates generally to detecting the presence of an amplifiedtarget sequence obtained by amplifying a sample containing at least onetarget sequence associated with a newborn disorder and a target-specificprimer designed according to the primer criteria provided herein.

In some embodiments, the one or more newborn disorders can include2-methyl-3-hydroxybutyric aciduria (2M3HBA); 2-methylbutyryl-CoAdehydrogenase (2 MBG); 3-methylglutaconic aciduria (3MGA); argininemia(ARG); defects of biopterin cofactor biosynthesis (BIOPT-BS); defects ofbiopterin cofactor regeneration (BIOPT-REG); carnitine acylcarnitinetranslocase (CACT); methylmalonic acidemia (CBL-C,D); citrullinemia typeII (CIT-II); carnitine palmitoyltransferase I (CPT-Ia); carnitinepalmitoyltransferase II (CPT-II); Dienoyl-CoA reductase (De-Red);Glutaric acidemia type II (GA-II); galactose epimerase (GALE);galactokinase (GALK); benign hyperphenylalaninemia (H-PHE);isobutyryl-CoA dehydrogenase (IBG); medium/short chain L-3-hydroxyacyl-CoA dehydrogenase (M/SCHAD); malonic acidemia (MAL); medium chainketoacyl-CoA thiolase (MCKAT); hypermethioninemia (MET); short chainacyl-CoA dehydrogenase (SCAD); Tyrosinemia type II (TYR-II); tyrosinemiatype III (TYR-III); Biotimidase (BIO); Cystic fibrosis (CF); Transferasedeficient galactosemia (GALT); Sickle-C disease (HB S/C); Congenitaladrenal hyperplasia (CAH); Congenital hypothyroidism (CH); Sickle cellanemia (HB S/S); S-βeta thalassemia (HB S/A); (SCID) Severe CombinedImmunodeficiency; 5-oxoprolinuria (pyroglutamic aciduria) (5-OXO);Glucose 6 phosphate dehydrogenase (G6PD); Nonketotic hyperglycinemia(NKH); Carbamoylphosphate synthetase (CPS);Hyperammonemia/ornithinemia/citrullinemia (Ornithine transporter defect)(HHH); Prolinemia (PRO); Ethylmalonic encephalopathy (EMA); Humanimmunodeficiency virus (HIV); Toxoplasmosis (TOXO); 3-Methylcrotonyl-CoAcarboxylase (3-MCC); Carnitine uptake defect (CUD); Long-chainL-3-hydroxyacyl-CoA dehydrogenase (LCHAD);Phenylketonuria/Hyperphenylalaninemia (PKU); Argininosuccinate aciduria(ASA); Glutaric acidemia type 1 (GA-1); Medium-chain acyl-CoAdehydrogenase (MCAD); Propionic acidemia (Propionyl-CoA carboxylase)(PROP); Beta ketothiolase (mitochondrial acetoacetyl-CoA thiolase;short-chain ketoacyl thiolase; T2) (BKT); Homocystinuria (cystathioninebeta synthase) (HCY); Multiple carboxylase (Holocarboxylase synthetase)(MCD); Trifunctional protein deficiency (TFP); Methylmalonic academia(Vitamin B12 Disorders) (CBL A,B); 3-Hydroxy 3-methylglutaric aciduria(3-Hydroxy 3-methylglutaryl-CoA lyase) (HMG); Maple syrup urine disease(branched-chain ketoacid dehydrogenase) (MSUD); Tyrosinemia Type 1(TYR-1); Citrullinemia type I (Argininosuccinate synthetase) (CIT I);Isovaleric acidemia (Isovaleryl-CoA dehydrogenase) (IVA); MethylmalonicAcidemia (methylmalonyl-CoA mutase) (MUT); and very long-chain acyl-CoAdehydrogenase (VLCAD).

In some embodiments, the disclosure relates generally to target-specificprimers for the detection of newborn screening disorders. In someembodiments, target-specific primers for newborn disorders including thedisorders provided above can be prepared using the primer criteriadisclosed herein. In some embodiments, the disclosure relates generallyto detecting a newborn disorder by contacting a sample that may containone or more target sequences for one or more newborn disorders andamplifying the one or more target sequences in the sample, therebyobtaining at least one amplified target sequence associated with atleast one newborn disorder. In some embodiments, a plurality oftarget-specific primers can be designed to amplify a plurality of targetsequences from a sample, thereby providing a means to detect a pluralityof newborn disorders optionally, in a single method or procedure. Insome embodiments, target-specific primers designed to amplify aplurality of amplified target sequences associated with one or morenewborn disorders can be pooled and provided as a newborn screeningpanel.

In some embodiments, target sequences or amplified target sequences aredirected to nucleic acids obtained from a forensic sample. In oneembodiment, forensic samples can include nucleic acids obtained from acrime scene, nucleic acids obtained from a missing persons DNA database,nucleic acids obtained from a laboratory associated with a forensicinvestigation or include forensic samples obtained by law enforcementagencies, one or more military services or any such personnel. In someembodiments, target sequences can be present in one or more bodilyfluids including but not limited to, blood, sputum, plasma, semen, urineand serum. In some embodiments, target sequences can be obtained fromhair, skin, tissue samples, autopsy or remains of a victim. In someembodiments, nucleic acids including one or more target sequences can beobtained from a deceased animal or human. In some embodiments, targetsequences can include nucleic acids obtained from non-human DNA such amicrobial, plant or entomological DNA. In some embodiments, targetsequences or amplified target sequences are directed to purposes ofhuman identification. In some embodiments, the disclosure relatesgenerally to methods for identifying a nucleic acid sample from ananimal, including a human. In some embodiments, the disclosure relatesgenerally to methods for identifying characteristics of a forensicsample. In some embodiments, the disclosure relates generally to humanidentification methods using one or more target-specific primersdisclosed herein or one or more target-specific primers prepared usingthe primer criteria outlined herein.

In one embodiment, a forensic or human identification sample containingat least one target sequence can be amplified using any one or more ofthe target-specific primers disclosed herein or using the primercriteria outlined herein. In some embodiments, a forensic or humanidentification sample containing one or more target sequences can beidentified by amplifying the at least one or more target sequences withany one or more target-specific primers provided in Tables 13 and 14.Table 13 provides a plurality of target-specific primers, provided asprimer pairs, directed to single nucleotide polymorphisms (SNPs)associated with human identification. Table 14 provides a plurality oftarget-specific primers, provided as primer pairs, directed to shorttandem repeats (STRs) associated with human identification. Anindividual inherits one copy of an STR from each parent, which may ormay not have similar repeat sizes. The number of repeats in STR markerscan be highly variable among individuals, which makes STRs effective forhuman identification purposes. In some embodiments, targets-specificprimers such as those provided in Table 14, or target-specific primersprepared as disclosed herein, that are directed to the gene amelogenin(AMG) can be used to determine the sex of the individual providing thesample. For example, primers to the amelogenin gene can be preparedusing the criteria disclosed herein that are specific, for example, tointron 1. Once a sample is amplified using such target-specific primers,the amplification product from a male sample versus a female sample willgenerally result in amplification products (amplified target sequences)that differ by in length by several nucleotides and therefore provides asimple method by which to determine the sex of the individual providingthe sample.

In one embodiment, a sample containing one or more target sequences canbe amplified using any one or more of the target-specific primersdisclosed herein. In another embodiment, amplified target sequencesobtained using the methods (and associated compositions, systems,apparatuses and kits) disclosed herein, can be coupled to a downstreamprocess, such as but not limited to, nucleic acid sequencing. Forexample, once the nucleic acid sequence of an amplified target sequenceis known, the nucleic acid sequence can be compared to one or morereference samples such as Hg19 genome. The Hg19 genome is commonly usedin the genomics field as a reference genome sample for humans. In someembodiments, a sample suspected of containing one or more SNPs and/orSTRs can be identified by amplifying the sample suspected of containingthe SNP or STR with any one or more of the target-specific primersprovided in Tables 13 and 14. Consequently, the output from theamplification procedure can be optionally analyzed for example bynucleic acid sequencing to determine if the expected amplificationproduct based on the target-specific primers is present in theamplification output. The identification of an appropriate SNP or STRamplification product can in some instances provide additionalinformation regarding the source of the sample or it characteristics(e.g., a male or female sample or a sample of particular ancestralorigin).

It is envisaged that one of ordinary skill in the art can readilyprepare one or more target-specific primers using the primer criteriadisclosed herein without undue experimentation. It is also envisagedthat one of ordinary skill in the art can readily prepare one or moretarget-specific primers using the criteria disclosed herein, to identifyat least one medically relevant polymorphism. In some instances, amedically relevant polymorphism can be used in forensic or humanidentification purposes. Generally, a medically relevant polymorphismincludes a polymorphism that is associated with at least one diseasestate in multiple populations (e.g., a European Caucasian population).In some embodiments, a medically relevant polymorphism includes any oneor more of the polymorphisms outlined below.

Polymorphism Caucasian MAF Chromosome Gene Disease Associated withPolymorphism rs1137101 0.449 1p31 LEPR Obesity, Insulin Resistance, Non-Hodgkin's lymphoma rs486907 0.408 1q25 RNASEL Prostate cancer rs10420310.208 2p24 APOB Cardiovascular disease, Dislipidemia rs231775 0.379 2q33CTLA4 Multiple Sclerosis, Autoimmune Disease rs5186 0.348 3q21 AGTR1Metabolic syndrome, Aortic aneurism, Left-ventricular hypertrophy rs62800.35 3q13.3 DRD3 Schizophrenia rs1693482 0.477 4q21 ADH1C Alcoholdependence, Coronary heart disease rs1799883 0.373 4q28 FABP2 Metabolicsyndrome, Type 2 diabetes rs4444903 0.392 4q25 EGF Cancer rs4961 0.2084p16.3 ADD1 Hypertension, Coronary artery disease rs1042714 0.467 5q31ADRB2 Obesity, COPD rs351855 0.283 5q35.1 FGFR4 Cancer rs5370 0.242 6p24EDN1 Asthma, sleep apnea rs6296 0.322 6q13 HTR1B Substance abusers2227983 0.25 7p12.3 EGFR Cancer rs213950 0.492 7q31.2 CFTR Cysticfibrosis rs7493 0.237 7q21.3 PON2 Myocardial infarction rs328 0.273 8p22LPL Left ventricular hypertrophy rs2383206 0.475 9p21 Coronary arterydisease rs1800861 0.25 10q11.2 RET Hirschsprung disease, Thyroid cancerrs1801253 0.283 10q24 ADRB1 Insulin Resistance rs2227564 0.341 10q24PLAU Alzheimer's disease, Asthma rs1799750 0.433 11q22.3 MMP1Endometriosis, Osteolysis, Rheumatoid arthritis rs1063856 0.342 12p13.3VWF Hypertension rs6313 0.438 13q14 HTR2A Psychiatric disordersrs2236225 0.396 14q24 MTHFD1 Neural tube defects rs1800588 0.333 15q21LIPC Coronary artery disease rs243865 0.198 16q13 MMP2 Cancer rs46730.342 16q24 CYBA Coronary artery disease rs708272 0.478 16q21 CETPCoronary artery disease rs1800012 0.188 17q21.3 COL1A1 Osteoporosisrs4291 0.354 17q23 ACE Depression, Alzheimer's disease rs4792311 0.33117p11 ELAC2 Prostate cancer rs16430 0.37 18p11.3 ENOSF1/TYMS Cancerrs601338 0.391 19q13.3 FUT2 Infection susceptibility rs688 0.45 19p13.2LDLR Alzheimer's disease, Coronary artery disease rs7121 0.458 20q13.3GNAS Obesity, Cancer rs234706 0.333 21q22 CBS Oral cleft defects rs46800.483 22q11.21 COMT Schizophrenia, ADHD AMG 0.5 Xp22.3 AMG Sex Marker

In some embodiments, a medically relevant polymorphism can be presentwithin a single exon of the corresponding disease associated gene. Insome embodiments, the disclosure relates generally to the selectiveamplification of at least one target sequence in a sample and thedetection and/or identification of a medically relevant polymorphism. Insome embodiments, the disclosure relates generally to the selectiveamplification of at least one target sequence in a sample and thedetection and/or identification of a SNP or STR. In some embodiments,amplified target sequences can be generated by amplifying a sample withone or more target-specific primers of Tables 13 or 14. In someembodiments, the amplified target sequences can include any one or moreof the amplified target sequences generated at the genomic coordinatesprovided in the polymorphism table above. In one embodiment, anamplified target sequence can be prepared using one or more of thetarget-specific primers from Tables 13 or 14. In some embodiments, anyone or more target-specific primers corresponding to SEQ ID NOs:50354-50451 can be used to selectively amplify at least one targetsequence present in a sample. In some embodiments, at least onetarget-specific primer selected from SEQ ID NOs: 50354-50451 can be usedto amplify a target sequence from a sample for the purposes of forensicor human identification.

In some embodiments, target-specific primers directed to humanidentification can be selected from any one or more of thetarget-specific primers provided in Tables 13 or 14. In someembodiments, the target-specific primers from Tables 13 or 14 caninclude 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 22, 25, 27, 30, 35, 38,40, 42, 45 or more target-specific primers. In some embodiments, atleast one of the target-specific primers associated with humanidentification is at least 90% identical to at least one of thetarget-specific primers provided in Tables 13 or 14. In someembodiments, at least one of the target-specific primers associated withhuman identification is complementary across its entire length to atleast one target sequence in a sample. In some embodiments, at least oneof the target-specific primers associated with human identificationincludes a non-cleavable nucleotide at the 3′ end. In some embodiments,the non-cleavable nucleotide at the 3′ end includes the terminal 3′nucleotide.

In some embodiments, the disclosure relates generally to methods (andassociated compositions, systems, apparatuses and kits) for reducing theformation of amplification artifacts in a multiplex PCR. In someembodiments, primer-dimers or non-specific amplification products areobtained in lower number or yield as compared to standard multiplex PCRof the prior art. In some embodiments, the reduction in amplificationartifacts is in part, governed by the use of target-specific primerpairs in the multiplex PCR reaction. In one embodiment, the number oftarget-specific primer pairs in the multiplex PCR reaction can begreater than 1000, 3000, 5000, 10000, 12000, or more. In someembodiments, the disclosure relates generally to methods (and associatedcompositions, systems, apparatuses and kits) for performing multiplexPCR using target-specific primers that contain a cleavable group. In oneembodiment, target-specific primers containing a cleavable group caninclude one or more cleavable moieties per primer of each primer pair.In some embodiments, a target-specific primer containing a cleavablegroup includes an nucleotide neither normally present in a non-diseasedsample nor native to the population of nucleic acids undergoingmultiplex PCR. For example, a target-specific primer can include one ormore non-native nucleic acid molecules such as, but not limited tothymine dimers, 8-oxo-2′-deoxyguanosine, inosine, deoxyuridine,bromodeoxyuridine, apurinic nucleotides, and the like.

In some embodiments, the disclosed methods (and associated compositions,systems, etc.) involve performing a primary amplification of targetsequences from a population of nucleic acids, optionally usingtarget-specific primers. In some embodiments, the disclosed methodsinvolve amplifying target sequences using target-specific forward andreverse primer pairs. The target-specific forward and reverse primerpairs can optionally include one or more intron-specific and/or exonspecific forward and reverse primer pairs. In some embodiments, eachprimer pair is directed to a single or discrete exon. In someembodiments, the disclosed methods involve amplifying target sequencesusing exon-specific forward and reverse primer pairs containing at leastone cleavable group. In some embodiments, the target-specific forwardand reverse primer pairs contain a uracil nucleotide as the one or morecleavable groups. In one embodiment, a target-specific primer pair caninclude a uracil nucleotide in each of the forward and reverse primersof each primer pair. In one embodiment, a target-specific forward orreverse primer contains one, two, three or more uracil nucleotides. Insome embodiments, the disclosed methods involve amplifying at least 10,50, 100, 200, 500, 1000, 2000, 3000, 5000, 6000, 8000, 10000, 12000 ormore, target sequences from a population of nucleic acids having aplurality of target sequences using target-specific forward and reverseprimer pairs containing at least two uracil nucleotides.

In some embodiments, target-specific primers (including but not limitedto intron-specific and exon-specific primers, which can be forwardand/or reverse primers) can be designed de novo using algorithms thatgenerate oligonucleotide sequences according to specified designcriteria. For example, the primers may be selected according to any oneor more of criteria specified herein. In some embodiments, one or moreof the target-specific primers are selected or designed to satisfy anyone or more of the following criteria: (1) inclusion of two or moremodified nucleotides within the primer sequence, at least one of whichis included near or at the termini of the primer and at least one ofwhich is included at, or about the center nucleotide position of theprimer sequence; (2) primer length of about 15 to about 40 bases inlength; (3) T_(m) of from about 60° C. to about 70° C.; (4) lowcross-reactivity with non-target sequences present in the target genomeor sample of interest; (5) for each primer in a given reaction, thesequence of at least the first four nucleotides (going from 3′ to 5′direction) are not complementary to any sequence within any other primerpresent in the same reaction; and (6) no amplicon includes anyconsecutive stretch of at least 5 nucleotides that is complementary toany sequence within any other amplicon.

In some embodiments, the target-specific primers include one or moreprimer pairs designed to amplify target sequences from the sample thatare about 100 base pairs to about 500 base pairs in length. In someembodiments, the target-specific primers include a plurality of primerpairs designed to amplify target sequences, where the amplified targetsequences are predicted to vary in length from each other by no morethan 50%, typically no more than 25%, even more typically by no morethan 10%, or 5%. For example, if one target-specific primer pair isselected or predicted to amplify a product that is 100 nucleotides inlength, then other primer pairs are selected or predicted to amplifyproducts that are between 50-150 nucleotides in length, typicallybetween 75-125 nucleotides in length, even more typically between 90-110nucleotides, or 95-105 nucleotides, or 99-101 nucleotides in length.

In some embodiments, at least one primer pair in the amplificationreaction is not designed de novo according to any predeterminedselection criteria. For example, at least one primer pair can be anoligonucleotide sequence selected or generated at random, or previouslyselected or generated for other applications. In one exemplaryembodiment, the amplification reaction can include at least one primerpair selected from the TaqMan® probe reagents (Roche Molecular Systems).The TaqMan® reagents include labeled probes and can be useful, interalia, for measuring the amount of target sequence present in the sample,optionally in real time. Some examples of TaqMan technology aredisclosed in U.S. Pat. Nos. 5,210,015, 5,487,972, 5,804,375, 6,214,979,7,141,377 and 7,445,900, hereby incorporated by reference in theirentireties.

In some embodiments, at least one primer within the amplificationreaction can be labeled, for example with an optically detectable label,to facilitate a particular application of interest. For example,labeling may facilitate quantification of target template and/oramplification product, isolation of the target template and/oramplification, product, and the like.

In some embodiments, one or more of the primers within the amplificationreaction can be useful in genotyping of a nucleic acid sample.

In some embodiments, the target-specific primers can be provided as aset of target-specific primer pairs in a single amplification vessel. Insome embodiments, the target-specific primers can be provided in one ormore aliquots of target-specific primer pairs that can be pooled priorto performing the multiplex PCR reaction in a single amplificationvessel or reaction chamber. In one embodiment, the target-specificprimers can be provided as a pool of target-specific forward primers anda separate pool of target-specific reverse primers. In anotherembodiment, target-specific primer pairs can be pooled into subsets suchas non-overlapping target-specific primer pairs. In some embodiments,the pool of target-specific primer pairs can be provided in a singlereaction chamber or microwell, for example on a PCR plate to performmultiplex PCR using a thermocycler. In some embodiments, thetarget-specific forward and reverse primer pairs can be substantiallycomplementary to the target sequences.

In some embodiments, the method of performing multiplex PCRamplification includes contacting a plurality of target-specific primerpairs having a forward and reverse primer, with a population of targetsequences to form a plurality of template/primer duplexes; adding a DNApolymerase and a mixture of dNTPs to the plurality of template/primerduplexes for sufficient time and at sufficient temperature to extendeither (or both) the forward or reverse primer in each target-specificprimer pair via template-dependent synthesis thereby generating aplurality of extended primer product/template duplexes; denaturing theextended primer product/template duplexes; annealing to the extendedprimer product the complementary primer from the target-specific primerpair; and extending the annealed primer in the presence of a DNApolymerase and dNTPs to form a plurality of target-specificdouble-stranded nucleic acid molecules. In some embodiments, the stepsof the amplification PCR method can be performed in any order. In someinstances, the methods disclosed herein can be further optimized toremove one or more steps and still obtain sufficient amplified targetsequences to be used in a variety of downstream processes. For example,the number of purification or clean-up steps can be modified to includemore or less steps than disclose herein, providing the amplified targetsequences are generated in sufficient yield.

In some embodiments, the target-specific primer pairs do not contain acommon extension (tail) at the 3′ or 5′ end of the primer. In anotherembodiment, the target-specific primers do not contain a Tag oruniversal sequence. In some embodiments, the target-specific primerpairs are designed to eliminate or reduce interactions that promote theformation of non-specific amplification.

In one embodiment, the target-specific primer pairs comprise at leastone cleavable group per forward and reverse target-specific primer. Inone embodiment, the cleavable group can be a uracil nucleotide. In oneembodiment, the target-specific primer pairs are partially orsubstantially removed after generation of the amplified target sequence.In one embodiment, the removal can include enzymatic, heat or alkalitreatment of the target-specific primer pairs as part of the amplifiedtarget sequence. In some embodiments, the amplified target sequences arefurther treated to form blunt-ended amplification products, referred toherein as, blunt-ended amplified target sequences.

In some embodiments, any one or more of the target-specific primersdisclosed in the methods, compositions, kits, systems and apparatusesmay be designed using the following primer selection criteria.

There is a need for new methods, computer readable media, and systemsfor identifying or designing products or kits that use PCR to enrich oneor more genomic regions of interest (which may be, for example,cumulative regions of 1 kb to 1 Mb) for subsequent sequencing.

There is a need for new methods, computer readable media, and systemsfor identifying or designing products or kits including primers orassays that maximize coverage of one or more genomic regions or targetsof interest while minimizing one or more of off-target hybridization, anumber of primers, and a number of primer pools.

In accordance with the teachings and principles embodied in thisapplication, new methods, computer readable media, and systems areprovided that identify or design products or kits that use PCR to enrichone or more genomic regions or targets of interest for subsequentsequencing and/or that include primers or assays that maximize coverageof one or more genomic regions or targets of interest while minimizingone or more of off-target hybridization, a number of primers, and anumber of primer pools.

FIG. 17 illustrates a system for designing primers or assays accordingto an exemplary embodiment. The system includes a data receiving module1701, a primer providing module 1702, a scoring (in silico PCR) module1703, a scoring (SNP overlap) module 1704, a filtering module 1705, apooling module 1706, and a reporting module 1707. The system alsoincludes a database 1708, which may include data regarding geneticannotations, SNP-related data, or other genetic data such asidentification of a repeat, chromosome, position, direction, etc., forexample, or any other type of information that could be related to agenomic region or target of interest, and a database 1709, which mayinclude primer-related data such as a melting temperature (Tm), achromosome, a position, a direction, and SNP overlap information, etc.,for example, or any other type of information that could be related toprimers. The system may be implemented in or using one or more computersand/or servers using one or more software components, which may not beaccessible or released to customers who may be ordering custom primersor assays that may be designed using such a system. Customers may ordercustom primers or assays at least in part through a web-accessible dataportal by providing one or more genomic regions or targets of interestin any suitable format. In an exemplary embodiment, there is provided amethod performing steps including the general steps associated withmodules 1701-1707 and databases 1708 and 1709 (e.g., receiving data,providing primers, scoring primers and/or amplicons, filtering primersand/or amplicons, pooling primers and/or amplicons, reporting results,and querying databases).

FIG. 18 illustrates a system for designing primers or assays accordingto an exemplary embodiment. The system includes a target generatormodule, which may generate one or more coordinate-based genomic regionsor targets of interest and which may query and/or receive informationfrom an annotation database (which may include data regarding geneticannotations, SNP-related data, or other genetic data such asidentification of a repeat, chromosome, position, direction, etc., forexample, as well as information regarding primers or any other type ofinformation that could be related to a genomic region or target ofinterest); a designing module, which may design one or more primers orassays and determine and/or apply various scoring and filteringprocedures for the primers or assays and which may perform variousquality control procedures; a loader module, which may load the primersor assays and/or related information (such as quality control results,for example) to a primer database (which may be in communication with orcomprised within the annotation database and which may includeprimer-related data such as a melting temperature (Tm), a chromosome, aposition, a direction, and SNP overlap information, etc., for example,or any other type of information that could be related to primers); aSNP overlap/repeat overlap module; a driver module; a tiler module,which may determine a subset of amplicons or tiles maximizing a coverageof a genomic region or target of interest; a pooler module, which maydetermine a pooling of the amplicons or tiles into one or more pools ofamplicons; and a report generator module. The system may be implementedin or using one or more computers and/or servers using one or moresoftware components, which may not be accessible or released tocustomers who may be ordering custom primers or assays that may bedesigned using such a system. Customers may order custom primers orassays at least in part through a web-accessible data portal byproviding one or more genomic regions or targets of interest in anysuitable format. In an exemplary embodiment, there is provided a methodperforming steps including the general steps associated with thesemodules and databases.

FIG. 19 illustrates an amplicon sequence including an insert sequencesurrounded by a pair of primers designed according to an exemplaryembodiment. The amplicon may include a forward primer and a reverseprimer surrounding the insert sequence. The two primers may togetherform an assay, which may be customized and ordered. The primer componentof an amplicon may be a copy of a spiked-in primer, rather than theunderlying sample, and one or more inserts may be selected to cover thetarget.

FIG. 20 illustrates PCR amplification of an amplicon sequence (which maybe referred to as “tile” herein) including an insert surrounded by apair of primers designed according to an exemplary embodiment. Shown aredenaturation, annealing, and elongation steps ultimately leading toexponential growth of the amplicon.

FIG. 21 (Part 1-Part III) illustrates a set of candidate amplicons for agiven target region, each including an insert surrounded by a pair ofprimers, for tiling and pooling according to an exemplary embodiment.The dotted lines indicate the boundaries of a target region (onchromosome 19 in this example). There are 112 candidate amplicons forcovering the target region in this example, but the number of candidateamplicons could of course be different, including much lower or muchhigher, and may be selected by taking into account computationalresources, the length of the target region, and any other relevantfactor.

According to various exemplary embodiments, there are provided methodsfor designing primers using a design pipeline that allows design ofoligonucleotide primers across genomic areas of interest whileincorporating various design criteria and considerations includingamplicon size, primer composition, potential off-target hybridization,and SNP overlap of the primers. In an embodiment, the design pipelineincludes several functional modules that may be sequentially executed asdiscussed next.

First, in an embodiment, a sequence retrieval module may be configuredto retrieve sequences based on instructions of an operator regarding afinal product desired by a customer. The operator may request a designof primer pairs for genomic regions which may be specified by chromosomeand genome coordinates or by a gene symbol designator. In the lattercase, the sequence retrieval module may retrieve the sequence based onthe exon coordinates. The operator may also specify whether to include a5′ UTR sequence (untranslated sequence).

Second, in an embodiment, an assay design module may be configured todesign primer pairs using a design engine, which may be a public toolsuch as Primer3 or another primer design software that can generateprimer pairs across the entire sequence regions retrieved by thesequence retrieval module, for example. The primers pairs may beselected to tile densely across the nucleotide sequence. The primerdesign may be based on various parameters, including: (1) the meltingtemperature of the primer (which may be calculated using the nearestneighbor algorithm set forth in John SantaLucia, Jr., “A unified view ofpolymer, dumbbell, and oligonucleotide DNA nearest-neighborthermodynamics,” Proc. Natl. Acad. Sci. USA, vol. 95, 1460-1465 (1998),the contents of which is incorporated by reference herein in itsentirety), (2) the primer composition (e.g., nucleotide composition suchas GC content may be determined and filtered and penalized by thesoftware, as may be primer hairpin formation, composition of the GCcontent in the 3′ end of primer, and specific parameters that may beevaluated are stretches of homopolymeric nucleotides, hairpin formation,GC content, and amplicon size), (3) scores of forward primer, reverseprimer and amplicon (the scores may be added up to obtain a probe setscore, and the score may reflect how close the amplicon confirms withthe intended parameters), and (4) conversion of some of the T's to U's(T's may be placed such that the predicted Tm of the T delimitedfragments of a primer have a minimum average Tm.)

Third, in an embodiment, a primer mapping module may be configured touse a mapping software (e.g., e-PCR(NCBI), see Rotmistrovsky et al., “Aweb server for performing electronic PCR,” Nucleic Acids Research, vol.32, W108-W112 (2004), and Schuler, “Sequence Mapping by Electronic PCR,”Genome Research, vol. 7, 541-550 (1997), which are both incorporated byreference herein in their entirety, or other similar software) to mapprimers to a genome. The primers mapping may be scored using a mismatchmatrix. In an embodiment, a perfect match may receive a score of 0, andmismatched primers may receive a score of greater than 0. The mismatchmatrix takes the position of the mismatch and the nature of the mismatchinto account. For example, the mismatch matrix may assign a mismatchscore to every combination of a particular motif (e.g., AA, AC, AG, CA,CC, CT, GA, GG, GT, TC, TG, TT, A-, C-, G-, T-, -A, -C, -G, and -T,where ‘-’ denotes an ambiguous base or gap) with a particular position(e.g., base at 3′ end, second base from 3′ end, third base from 3′ end,third base from 5′ end, second base from 5′ end, base at 5′ end, andpositions therebetween), which may be derived empirically and may beselected to reflect that mismatches closer to the 3′ end tend to weakerPCR reactions more than mismatches closer to the 5′ end and maytherefore be generally larger. The mismatch scores for motifs with anambiguous base or gap may be assigned an average of scores of othermotifs consistent therewith (e.g., A- may be assigned an average of thescores of AA, AC, and AG). Based on the number of hits with a certainscore threshold, an amplicon cost may be calculated.

Fourth, in an embodiment, a SNP module may be configured to determineunderlying SNPs and repeat regions: SNPs may be mapped to the primersand based on the distance of a SNP from the 3′ end, primers may befiltered as potential candidates. Similarly, if a primer overlaps to acertain percentage with a repeat region, the primer might be filtered.

Fifth, in an embodiment, a tiler module may be configured to use afunction based on the amplicon cost (see primer mapping) and the numberof primers necessary to select a set of primers covering the targetwhile ensuring that selection of tiling primers for a target isindependent of other targets that may be in a customer's request so thatthe same set of primers for a target will be selected whether thecustomer requested only that target or additional targets and whetheramplicons are to help cover on that target or additional targets.

Sixth, in an embodiment, a pooler module may be configured to use apooling algorithm that prevents amplicon overlaps, and ensures that theaverage number of primers in a pool does not deviate by more than apreset value.

FIG. 22 illustrates a method according to an exemplary embodiment. Instep 2201, a module or other hardware and/or software component receivesone or more genomic regions or sequences of interest. In step 2202, amodule or other hardware and/or software component determines one ormore target sequences for the received one or more genomic regions orsequences of interest. In step 2203, a module or other hardware and/orsoftware component provides one or more primer pairs for each of thedetermined one or more target sequences. In step 2204, a module or otherhardware and/or software component scores the one or more primer pairs,wherein the scoring comprises a penalty based on the performance of insilico PCR for the one or more primer pairs, and wherein the scoringfurther comprises an analysis of SNP overlap for the one or more primerpairs. In step 2205, a module or other hardware and/or softwarecomponent filters the one or more primer pairs based on a plurality offactors, including at least the penalty and the analysis of SNP overlap,to identify a filtered set of primer pairs corresponding to one or morecandidate amplicon sequences for the one or more genomic regions orsequences of interest.

According to an exemplary embodiment, there is provided a method,comprising: (1) receiving one or more genomic regions or sequences ofinterest; (2) determining one or more target sequences for the receivedone or more genomic regions or sequences of interest; (3) providing oneor more primer pairs for each of the determined one or more targetsequences; (4) scoring the one or more primer pairs, wherein the scoringcomprises a penalty based on the performance of in silico PCR for theone or more primer pairs, and wherein the scoring further comprises ananalysis of SNP overlap for the one or more primer pairs; and (5)filtering the one or more primer pairs based on a plurality of factors,including at least the penalty and the analysis of SNP overlap, toidentify a filtered set of primer pairs corresponding to one or morecandidate amplicon sequences for the one or more genomic regions orsequences of interest.

In various embodiments, receiving one or more genomic regions orsequences of interest may comprise receiving a list of one or more genesymbols or identifiers. Receiving one or more genomic regions orsequences of interest may comprise receiving a list of one or moregenomic coordinates or other genomic location identifiers. Receiving oneor more genomic regions or sequences of interest may comprise receivinga list of one or more BED coordinates.

In various embodiments, determining one or more target sequences maycomprise determining one or more exons or coding regions that correspondto each of the one or more genomic regions or sequences of interest.Determining one or more target sequences may comprise querying anamplicon or other genomic sequence database for a presence therein ofthe one or more genomic regions or sequences of interest and informationrelated thereto.

In various embodiments, providing one or more primer pairs may comprisedesigning one or more primer pairs. Providing one or more primer pairsmay comprise querying an amplicon or other genomic sequence database fora presence therein of the one or more genomic regions or sequences ofinterest or of the one or more primer pairs and information relatedthereto.

In various embodiments, the performance of in silico PCR may compriseperforming in silico PCR against a reference or previously sequencedgenome of any species. The performance of in silico PCR may compriseperforming in silico PCR against an hg19 reference genome. Theperformance of in silico PCR against a reference genome may comprisedetermining a number of off-target hybridizations for each of the one ormore primer pairs. The performance of in silico PCR against a referencegenome may comprise determining a worst case attribute or score for eachof the one or more primer pairs. The performance of in silico PCR maycomprise determining one or more genomic coordinates for each of the oneor more primer pairs. The performance of in silico PCR may comprisedetermining one or more predicted amplicon sequences for each of the oneor more primer pairs. The performance of in silico PCR may comprisequerying an amplicon or other genomic sequence database for a presencetherein of the one or more genomic regions or sequences of interest orof in silico PCR results for the one or more primer pairs andinformation related thereto.

In various embodiments, the analysis of SNP overlap may comprisedetermining a SNP class for each of the one or more primer pairs. Theanalysis of SNP overlap may comprise querying an amplicon or othergenomic sequence database for a presence therein of the one or moregenomic regions or sequences of interest or of SNP overlap results forthe one or more primer pairs and information related thereto.

In various embodiments, the plurality of factors may include one or moreof an indication of forward SNP overlap, an indication of a reverse SNPoverlap, an indication of a frequency of forward repeats, an indicationof a frequency of reverse repeats, an indication of an off-targethybridization of each of the one or more primer pairs, and a compositionof each of the one or more primer pairs. The plurality of factors mayinclude one or more of a forward triplet factor, a reverse tripletfactor, a forward A run factor, a reverse A run factor, a forward C runfactor, a reverse C run factor, a forward G run factor, a reverse G runfactor, a forward T run factor, and a reverse T run factor. Theplurality of factors may include one or more of an indication of anextent to which each of the one or more primer pairs includes one ormore homopolymers. The plurality of factors may include an indication ofan extent to which each of the one or more primer pairs includes one ormore repeating sequences. The plurality of factors may include a lengthof the one or more primer pairs, wherein a score for the one or moreprimer pairs decreases as the length gets shorter than a minimal lengththreshold and decreases as the length gets longer than a maximal lengththreshold. The plurality of factors may include a maximal number of agiven base in the one or more primer pairs, wherein a score for the oneor more primer pairs decreases as the number of instances of the givenbase exceeds a maximal base inclusion threshold. The plurality offactors may include a maximal number of contiguous instances of a givenbase, wherein a score for the one or more primer pairs decreases as thenumber of contiguous instances of the given base exceeds a maximalcontiguous base inclusion threshold.

The plurality of factors may include a maximal percentage of a set oftwo given bases, wherein a score for the one or more primer pairsdecreases as the percentage of the two given bases increases. Theplurality of factors may include a maximal percentage of G and C bases,wherein a score for the one or more primer pairs decreases as thepercentage of G and C bases increases. The plurality of factors mayinclude a deviation of a predicted melting temperature for the one ormore primer pairs relative to minimal and maximal melting temperaturethresholds. The plurality of factors may include a number ofprimer-dimer inclusions for the one or more primer pairs. The pluralityof factors may include a level of local complementarity for the one ormore primer pairs. The plurality of factors may include an indication ofa complexity level of each of the one or more primer pairs. Theplurality of factors may include an indication of SNP overlap of each ofthe one or more primer

In various embodiments, the method may comprise selecting a subset ofthe one or more candidate amplicon sequences that substantially coversthe one or more genomic regions or sequences of interest whileminimizing a cost function associated with the candidate ampliconsequences. Minimizing the cost function may include generating anoverlap graph comprising a source vertex, one or more amplicon vertices,and a sink vertex.

In various embodiments, the method may comprise assembling the primerpairs in the filtered set of primer pairs that correspond with theselected subset of the one or more candidate amplicon sequences into aplurality of separate pools of primer pairs. Assembling the primer pairsmay include limiting an inclusion of one or more primer pairs in thefiltered set of primer pairs that correspond with the selected subset ofthe one or more candidate amplicon sequences into a given pool based atleast on a minimal threshold distance between amplicon sequences in thegiven pool. The minimal threshold distance may be between about 5 basepairs and about 100 base pairs, or between about 15 base pairs and about90 base pairs, or between about 25 base pairs and about 75 base pairs,or between about 40 base pairs and about 60 base pairs, for example. Insome embodiments, the minimum threshold distance between amplicons mayinclude any integer, including a negative one. For example, a value of 0can mean that any two amplicons are allowed to “touch,” and a value of−8 can mean that any two amplicons can overlap by up to 8 bases.

In various embodiments, assembling the filtered set of primer pairs intoa plurality of separate pools of primer pairs may comprise splitting theprimer pairs between tubes so as to prevent amplicon overlap within anygiven tube. Assembling the primer pairs may include limiting aninclusion of one or more primer pairs in the filtered set of primerpairs that correspond with the selected subset of the one or morecandidate amplicon sequences into a given pool based at least on apre-determined amplicon capacity of the given pool. Assembling theprimer pairs may include limiting an inclusion of one or more primerpairs in the filtered set of primer pairs that correspond with theselected subset of the one or more candidate amplicon sequences into agiven pool based at least on an inequality relating a size of the givenpool with a product between a balance factor and a maximum value of thesizes of the separate pools.

In various embodiments, the method may comprise providing a reportreporting on any one or more element of information of data used orgenerated by any one or more of the receiving, providing, scoring,filtering, selecting, and assembling steps.

According to an exemplary embodiment, there is provided a non-transitorymachine-readable storage medium comprising instructions which, whenexecuted by a processor, cause the processor to perform a methodcomprising: (1) receiving one or more genomic regions or sequences ofinterest; (2) determining one or more target sequences for the receivedone or more genomic regions or sequences of interest; (3) providing oneor more primer pairs for each of the determined one or more targetsequences; (4) scoring the one or more primer pairs, wherein the scoringcomprises a penalty based on the performance of in silico PCR for theone or more primer pairs, and wherein the scoring further comprises ananalysis of SNP overlap for the one or more primer pairs; and (5)filtering the one or more primer pairs based on a plurality of factors,including at least the penalty and the analysis of SNP overlap, toidentify a filtered set of primer pairs corresponding to one or morecandidate amplicon sequences for the one or more genomic regions orsequences of interest.

In various embodiments, such a non-transitory machine-readable storagemedium may comprise instructions which, when executed by a processor,cause the processor to perform a method further comprising: (6)selecting a subset of the one or more candidate amplicon sequences thatsubstantially covers the one or more genomic regions or sequences ofinterest while minimizing a cost function associated with the candidateamplicon sequences; and (7) assembling the primer pairs in the filteredset of primer pairs that correspond with the selected subset of the oneor more candidate amplicon sequences into a plurality of separate poolsof primer pairs.

According to an exemplary embodiment, there is provided a system,comprising: (1) a machine-readable memory; and (2) a processorconfigured to execute machine-readable instructions, which, whenexecuted by the processor, cause the system to perform steps including:(a) receiving one or more genomic regions or sequences of interest; (b)determining one or more target sequences for the received one or moregenomic regions or sequences of interest; (c) providing one or moreprimer pairs for each of the determined one or more target sequences;(d) scoring the one or more primer pairs, wherein the scoring comprisesa penalty based on the performance of in silico PCR for the one or moreprimer pairs, and wherein the scoring further comprises an analysis ofSNP overlap for the one or more primer pairs; and (e) filtering the oneor more primer pairs based on a plurality of factors, including at leastthe penalty and the analysis of SNP overlap, to identify a filtered setof primer pairs corresponding to one or more candidate ampliconsequences for the one or more genomic regions or sequences of interest.

In various embodiments, the processor of such a system may further beconfigured to execute machine-readable instructions, which, whenexecuted by the processor, cause the system to perform steps including:(f) selecting a subset of the one or more candidate amplicon sequencesthat substantially covers the one or more genomic regions or sequencesof interest while minimizing a cost function associated with thecandidate amplicon sequences; and (g) assembling the primer pairs in thefiltered set of primer pairs that correspond with the selected subset ofthe one or more candidate amplicon sequences into a plurality ofseparate pools of primer pairs.

According to various exemplary embodiment, various parameters orcriteria may be used to select primers and/or amplicons.

In an embodiment, a forward SNP score may be used and may be given anumerical attribute/score of 1 if there is no SNP within a given lengthof base pairs of the forward primer (such as 4, for example) or anumerical attribute of 0 if there is one or more SNPs within a length of4 base pairs. In one embodiment, the forward SNP score may be given anumerical attribute/score of 1 if there is no SNP within a given lengthof base pairs from the 3′ end of the forward primer. In someembodiments, a SNP can include one or more SNPs found on UCSC's GenomeBrowser Web Page including but not limited to, the SNP reference tablereferred to as “dbSNP132 common”. An attribute/score of 1 may be aminimal attribute/score such that failure to achieve thatattribute/score would result in disqualification. The base lengththreshold for the attribute/score determination could be lower or higherthan 4, and could be 5, 6, 7, 8, 9, 10, 15, 20, for example, or moregenerally any positive integer larger than 4. The attribute/score couldbe other than binary and could be a more complex linear or non-linearfunction of the number of SNPs within the given length of base pairs.

In an embodiment, a reverse SNP score may be used and may be given anumerical attribute/score of 1 if there is no SNP within a given lengthof base pairs of the reverse primer (such as 4, for example) or anumerical attribute of 0 if there is one or more SNPs within a length of4 base pairs. In one embodiment, the reverse SNP score may be given anumerical attribute/score of 1 if there is no SNP within a given lengthof base pairs from the 3′ end of the reverse primer. In someembodiments, a SNP can include one or more SNPs found on UCSC's GenomeBrowser Web Page including but not limited to, the SNP reference tablereferred to as “dbSNP132 common”. An attribute/score of 1 may be aminimal attribute/score such that failure to achieve thatattribute/score would result in disqualification. The base lengththreshold for the attribute/score determination could be lower or higherthan 4, and could be 5, 6, 7, 8, 9, 10, 15, 20, for example, or moregenerally any positive integer larger than 4. The attribute/score couldbe other than binary and could be a more complex linear or non-linearfunction of the number of SNPs within the given length of base pairs.

In an embodiment, a forward repeat score may be used and may be given anumerical attribute/score of 1 if there is no repeat within a givenlength of base pairs of the forward primer (such as 4, for example) or anumerical attribute of 0 if there is one or more repeats within a lengthof 4 base pairs. In one embodiment, the forward repeat score may begiven a numerical attribute/score of 1 if there is less than 30% overlapof the forward primer with known repeats. In some embodiments, knownrepeats may include one or more repeats reported by UCSC's GenomeBrowser, for example repeat regions as provided by the repeat maskedhg19 genome from UCSC. An attribute/score of 1 may be a minimalattribute/score such that failure to achieve that attribute/score wouldresult in disqualification. The base length threshold for theattribute/score determination could be lower or higher than 4, and couldbe 5, 6, 7, 8, 9, 10, 15, 20, for example, or more generally anypositive integer larger than 4. The attribute/score could be other thanbinary and could be a more complex linear or non-linear function of thenumber of repeats within the given length of base pairs.

In an embodiment, a reverse repeat score may be used and may be given anumerical attribute/score of 1 if there is no repeat within a givenlength of base pairs of the reverse primer (such as 4, for example) or anumerical attribute of 0 if there is one or more repeats within a lengthof 4 base pairs. In one embodiment, the reverse repeat score may begiven a numerical attribute/score of 1 if there is less than 30% overlapof the reverse primer with known repeats. In some embodiments, knownrepeats may include one or more repeats reported by UCSC's GenomeBrowser, for example repeat regions as provided by the repeat maskedhg19 genome from UCSC. An attribute/score of 1 may be a minimalattribute/score such that failure to achieve that attribute/score wouldresult in disqualification. The base length threshold for theattribute/score determination could be lower or higher than 4, and couldbe 5, 6, 7, 8, 9, 10, 15, 20, for example, or more generally anypositive integer larger than 4. The attribute/score could be other thanbinary and could be a more complex linear or non-linear function of thenumber of repeats within the given length of base pairs.

In various embodiments, one or more of a forward triplet score, areverse triplet score, a forward A run score, a reverse A run score, aforward C run score, a reverse C run score, a forward G run score, areverse G run score, a forward T run score, and a reverse T run score,may be used and may be given a numerical attribute/score equal to thenumber of forward triplets, reverse triplets, forward A runs, reverse Aruns, forward C runs, reverse C runs, forward G runs, reverse G runs,forward T runs, and reverse T runs within the entire primer. Anattribute/score of 3 may be a maximal attribute/score for the tripletssuch that failure to remain at or below that attribute/score wouldresult in disqualification. An attribute/score of 5 may be a maximalattribute/score for the runs such that failure to remain at or belowthat attribute/score would result in disqualification. Theattribute/score could be other than binary and could be a more complexlinear or non-linear function of the number of triplets/runs.

In an embodiment, a length of the primers may be limited by a minimumprimer length threshold and a maximum primer length, and a length scorefor the primers may be set so as to decrease as the length gets shorterthan the minimum primer length threshold and to decrease as the lengthgets longer than the maximum primer length threshold. In an embodiment,the minimum primer length threshold may be 16. In other embodiments, theminimum primer length threshold may be 15, 14, 13, 12, 11, 10, 9, 8, 7,6, or 5, for example, and may also be 17, 18, 19, 20, 21, 22, 23, and24, for example. In an embodiment, the maximum primer length thresholdmay be 28. In other embodiments, the maximum primer length threshold maybe 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40, for example, andmay also be 27, 26, 25, 24, 23, 22, 21, and 20, for example. In anembodiment, the primer length criterion may be given a score of 1.0 ifthe length thresholds are satisfied, for example, and that score may godown to 0.0 as the primer length diverges from the minimum or maximumlength threshold. For example, if the maximum primer length thresholdwere set to 28, then the score could be set to 1.0 if the length doesnot exceed 28, to 0.7 if the length is 29, to 0.6 if the length is 30,to 0.5 if the length is 31, to 0.3 if the length is 32, to 0.1 if thelength is 33, and to 0.0 if the length is 34 or more. Theattribute/score could be scaled between values other than 0.0 and 1.0,of course, and the function defining how the score varies with anincrease difference relative to the threshold could be any other or morecomplex linear or non-linear function that does not lead to increases inscore for primer that further diverge from length thresholds.

In an embodiment, a number of G bases (or of A, C, or T bases) in theprimers may be limited by a maximum threshold, and corresponding scorefor the primers may be set so as to decrease as the number of G bases(or of A, C, or T bases) exceeds the maximum threshold. In anembodiment, the maximum threshold may be 3. In other embodiments, themaximum threshold may be 2, 4, 5, 6, 7, 8, 9, and 10, for example. In anembodiment, the number of G bases (or of A, C, or T bases) criterion maybe given a score of 1.0 if the maximum threshold is satisfied, forexample, and that score may go down to 0.0 as the number of G bases (orof A, C, or T bases) diverges from the maximum threshold. For example,if the maximum threshold were set to 4, then the score could be set to1.0 if the number of G bases (or of A, C, or T bases) does not exceed 4,to 0.9 if the number is 5, to 0.8 if the number is 6, to 0.6 if thenumber is 7, to 0.4 if the number is 8, to 0.2 if the number is 9, andto 0.0 if the number is 10 or more. The score could be scaled betweenvalues other than 0.0 and 1.0, of course, and the function defining howthe score varies with an increased difference between the number of Gbases (or of A, C, or T bases) and the maximum threshold could be anyother or more complex linear or non-linear function that does not leadto increases in score for primer that further diverge from the maximumthreshold.

In an embodiment, the numbers of contiguous and total matches in a loop(e.g., hairpin) in the primers may be limited by a maximum threshold,and corresponding scores for the primers may be set so as to decrease asthe numbers of contiguous and total matches in a loop exceed the maximumthreshold. In an embodiment, the maximum threshold for contiguousmatches may be 3 and the maximum threshold for total matches may be 5.In other embodiments, the maximum threshold for contiguous matches maybe 2, 4, 5, 6, 7, 8, 9, and 10, for example, and the maximum thresholdfor total matches may be 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15,for example. In an embodiment, the numbers of contiguous and totalmatches in a loop criteria may be given a score of 1.0 if the maximumthreshold is satisfied, for example, and that score may go down to 0.0as the number of the numbers of contiguous and total matches in a loopdiverge from the corresponding maximum threshold. For example, if themaximum threshold for contiguous matches were set to 3, then the scorecould be set to 1.0 if the number of contiguous matches does not exceed3, to 0.9 if the number is 4, to 0.7 if the number is 5, to 0.4 if thenumber is 6, to 0.2 if the number is 7, to 0.1 if the number is 8, andto 0.0 if the number is 9 or more. For example, if the maximum thresholdfor total matches were set to 5, then the score could be set to 1.0 ifthe number of total matches does not exceed 5, to 0.9 if the number is6, to 0.8 if the number is 7, to 0.6 if the number is 8, to 0.4 if thenumber is 9, to 0.2 if the number is 10, to 0.1 if the number is 11, andto 0.0 if the number is 12 or more. The scores could be scaled betweenvalues other than 0.0 and 1.0, of course, and the function defining howthe scores vary with an increased difference between the number ofcontiguous/total matches and the corresponding maximum thresholds couldbe any other or more complex linear or non-linear function that does notlead to increases in score for primer that further diverge from themaximum threshold.

In an embodiment, a number of G and C bases (or any two of the A, C, G,and T bases) in the last five bases of the primers may be limited by amaximum threshold, and corresponding score for the primers may be set soas to decrease as the number of G and C bases (or any two of the A, C,G, and T bases) exceeds the maximum threshold. In an embodiment, themaximum threshold may be 2. In other embodiments, the maximum thresholdmay be 3, 4, and 5, for example. In an embodiment, the number of G and Cbases (or any two of the A, C, G, and T bases) criterion may be given ascore of 1.0 if the maximum threshold is satisfied, for example, andthat score may go down to 0.0 as the number of G and C bases (or any twoof the A, C, G, and T bases) diverges from the maximum threshold. Forexample, if the maximum threshold were set to 2, then the score could beset to 1.0 if the number of G and C bases (or any two of the A, C, G,and T bases) does not exceed 2, to 0.8 if the number is 3, to 0.4 if thenumber is 4, and to 0.1 if the number is 5. The score could be scaledbetween values other than 0.0 and 1.0, of course, and the functiondefining how the score varies with an increased difference between thenumber of G and C bases (or any two of the A, C, G, and T bases) and themaximum threshold could be any other or more complex linear ornon-linear function that does not lead to increases in score for primerthat further diverge from the maximum threshold. In other embodiments,this criterion could consider the number of G and C bases (or any two ofthe A, C, G, and T bases) in a larger window of bases, such as in thelast six bases, the last seven bases, the last eight bases, etc., forexample.

In an embodiment, a percentage of G and C bases (or any two of the A, C,G, and T bases) in the primers may be limited by minimum and maximumthresholds, and corresponding score for the primers may be set so as todecrease as the percentage of G and C bases (or any two of the A, C, G,and T bases) diverges from the minimum or maximum threshold. In anembodiment, the minimum threshold may be 0.2 (20%) and the maximumthreshold may be 0.8 (80%). In other embodiments, the minimum thresholdmay be any percentage between about 0.2 (20%) and about 0.5 (50%) andthe maximum threshold may be any percentage between about 0.8 (80%) and0.5 (50%), for example. In an embodiment, the percentage of G and Cbases (or any two of the A, C, G, and T bases) criterion may be given ascore of 1.0 if the minimum and maximum thresholds are satisfied, forexample, and that score may go down to 0.0 if either of the thresholdsis not satisfied. The score could be scaled between values other than0.0 and 1.0, of course, and the function defining how the score varieswith an increased difference between the percentage of G and C bases (orany two of the A, C, G, and T bases) and the minimum or maximumthreshold could be any other or more complex linear or non-linearfunction that does not lead to increases in score for primer thatfurther diverge from the minimum or maximum threshold.

In an embodiment, a melting temperature (Tm) of the primers may belimited by minimum and maximum thresholds, and corresponding score forthe primers may be set so as to decrease as the melting temperaturediverges from the minimum or maximum threshold. In an embodiment, theminimum threshold may be 60 and the maximum threshold may be 67 with atarget melting temperature of 62. In other embodiments, the minimumthreshold may be a value between about 55 and about 65 and the maximumthreshold may be a value between about 62 and about 72, for example. Inan embodiment, the melting temperature criterion may be given a score of1.0 if the minimum and maximum thresholds are satisfied, for example,and that score may go down to 0.0 if either of the thresholds is notsatisfied. The score could be scaled between values other than 0.0 and1.0, of course, and the function defining how the score varies with anincreased difference between the melting temperature and the minimum ormaximum threshold could be any other or more complex linear ornon-linear function that does not lead to increases in score for primerthat further diverge from the minimum or maximum threshold. The meltingtemperature of a primer may be calculated using the teachings set forthin John SantaLucia, Jr., “A unified view of polymer, dumbbell, andoligonucleotide DNA nearest-neighbor thermodynamics,” Proc. Natl. Acad.Sci. USA, vol. 95, 1460-1465 (1998), the contents of which isincorporated by reference herein in its entirety.

In an embodiment, a primer-dimer propensity in the primers may belimited by a maximum threshold of contiguous primer-dimers at the 3′ endand a maximum threshold of total contiguous matches over the fulllength, and corresponding score for the primers may be set so as todecrease as the primer-dimer propensity diverges from the maximumthresholds. In an embodiment, the maximum threshold of contiguousprimer-dimers at the 3′ end may be 4 and the maximum threshold of totalcontiguous matches over the full length may be 8. In other embodiments,the maximum threshold of contiguous primer-dimers at the 3′ end may be avalue between about 2 and about 6 and the maximum threshold of totalcontiguous matches over the full length may be a value between about 4and 10, for example. In an embodiment, the primer-dimer propensitycriteria may be given a score of 1.0 if the threshold is satisfied, forexample, and that score may go down to 0.0 if the threshold is notsatisfied. The score could be scaled between values other than 0.0 and1.0, of course, and the function defining how the score varies with anincreased difference between the primer-dimer propensity and the maximumthreshold could be any other or more complex linear or non-linearfunction that does not lead to increases in score for primer thatfurther diverge from the maximum threshold.

In an embodiment, a percentage of G and C bases (or any two of the A, C,G, and T bases) in an amplicon sequence may be limited by minimum andmaximum thresholds, and corresponding score for the amplicons may be setso as to decrease as the percentage of G and C bases (or any two of theA, C, G, and T bases) diverges from the minimum or maximum threshold. Inan embodiment, the minimum threshold may be 0.0 (0%) and the maximumthreshold may be 1.0 (100%). In other embodiments, the minimum thresholdmay be any percentage between about 0.1 (10%) and about 0.25 (25%) andthe maximum threshold may be any percentage between about 0.75 (75%) and0.9 (90%), for example. In an embodiment, the percentage of G and Cbases (or any two of the A, C, G, and T bases) criterion may be given ascore of 1.0 if the minimum and maximum thresholds are satisfied, forexample, and that score may go down to 0.0 if either of the thresholdsis not satisfied. The score could be scaled between values other than0.0 and 1.0, of course, and the function defining how the score varieswith an increased difference between the percentage of G and C bases (orany two of the A, C, G, and T bases) and the minimum or maximumthreshold could be any other or more complex linear or non-linearfunction that does not lead to increases in score for amplicons thatfurther diverge from the minimum or maximum threshold.

In an embodiment, a length of the amplicons may be limited by a minimumamplicon length threshold and a maximum amplicon length, and a lengthscore for the amplicons may be set so as to decrease as the length getsshorter than the minimum amplicon length threshold and to decrease asthe length gets longer than the maximum amplicon length threshold. In anembodiment, the minimum amplicon length threshold may be 110. In otherembodiments, the minimum primer length threshold may be a value betweenabout 80 and about 140, for example. In an embodiment, the maximumamplicon length threshold may be 240. In other embodiments, the maximumamplicon length threshold may be a value between about 200 and about280, for example. In an embodiment, the amplicon length criterion may begiven a score of 1.0 if the length thresholds are satisfied and of 0.0if either is not satisfied. In another embodiment, that score may godown to 0.0 as the amplicon length diverges from the minimum or maximumlength threshold. For example, if the maximum amplicon length thresholdwere set to 240, then the score could be set to 1.0 if the length doesnot exceed 240, to 0.8 if the length is at least 250, to 0.6 if thelength is at least 260, to 0.4 if the length is at least 270, to 0.1 ifthe length is at least 280, and to 0.0 if the length is at least 290.The attribute/score could be scaled between values other than 0.0 and1.0, of course, and the function defining how the score varies with anincrease difference relative to the threshold could be any other or morecomplex linear or non-linear function that does not lead to increases inscore for amplicons that further diverge from length thresholds.

According to an exemplary embodiment, there is provided a method forselecting a subset (which may be referred to as “tiling” herein) ofamplicons (which may be referred to as “tiles” herein) from a pluralityof candidate amplicons for covering one or more specific desired (e.g.,customized) genomic regions or targets using one or more pools ofamplicons. The method may include receiving as input a set of one ormore targets and a set of candidate amplicons, and may includeoutputting as output a subset of the candidate amplicons and anassignment of each of the amplicons in the subset to a pool in whichthat amplicon can be multiplexed. Amplicons of any suitable sizes may beused. In an embodiment, an assay or primer design may accommodate 200 bpamplicons and 150 bp amplicons, for example, which may be especiallyuseful for certain challenging samples such as FFPE, for example. In anembodiment, an assay or primer design may be adapted to be compatiblewith one or more specific library kits, such as, for example, the IonAmpliSeq™ Library Kit 2.0.

According to an exemplary embodiment, there is provided a method fortiling and pooling, comprising (1) selecting a subset of amplicons(which may be referred to as “tiling” herein) from a set of inputamplicons such that the subset of amplicons (i) covers as much of eachtarget as do the amplicons in the set of input amplicons, (ii) has manyfewer amplicons than the set of input amplicons, and (iii) maximizes aquality of the amplicons; and (2) assigns each amplicon or tile in thesubset of amplicons or tiling to a pool so as to allow each pool to bemultiplexed.

FIG. 23 illustrates a method for tiling a plurality of amplicons for oneor more given targets according to an exemplary embodiment. In step2301, a module or other hardware and/or software component sorts the oneor more given targets by their start positions (or ensures that thegiven targets were already pre-sorted in such a manner when provided asinput). In step 2302, a module or other hardware and/or softwarecomponent sorts the amplicons by insert start (or ensures that theamplicons were already pre-sorted in such a manner when provided asinput). In step 2303, a module or other hardware and/or softwarecomponent merges overlapping targets present in the sorted one or moretargets. In step 2304, for each merged overlapping target, a module orother hardware and/or software component (i) determines what ampliconshave inserts overlapping the target and identifies such amplicons ascandidate amplicons, and (ii) determines tiles using a function of theone or more given targets and the candidate amplicons. In step 2305, amodule or other hardware and/or software component outputs thedetermined tiles. In some embodiments, targets may or may not be mergedahead of gathering any target amplicons, amplicons may be gathered forunmerged targets, and, if two targets share at least one amplicon, suchtwo targets may be merged together (and one of the two targets mayalready represent a set of merged input amplicons).

According to an exemplary embodiment, there is provided a method fortiling a plurality of amplicons for one or more given targets,comprising: (1) sorting the one or more given targets by their startpositions or ensuring that the given targets were already pre-sorted insuch a manner when provided as input; (2) sorting the amplicons byinsert start or ensuring that the amplicons were already pre-sorted insuch a manner when provided as input; (3) merging overlapping targetspresent in the sorted one or more targets; (4) for each mergedoverlapping target, (i) determining what amplicons have insertsoverlapping the target and identifying such amplicons as candidateamplicons and (ii) determining the tiles using a function of the one ormore given targets and the candidate amplicons; and (5) outputting thetiles.

FIG. 24 illustrates a method for determining tiles for one or more giventargets and candidate amplicons according to an exemplary embodiment. Instep 2401, a module or other hardware and/or software component createsan overlap graph of candidate inserts, the graph including a sourcevertex, one or more amplicon vertices, and a sink vertex along with oneor more edges linking vertices. In step 2402, a module or other hardwareand/or software component defines an edge cost function or uses adefined edge cost function. In step 2403, a module or other hardwareand/or software component finds a least cost path for such an edge costfunction from the source vertex to the sink vertex. In step 2404, amodule or other hardware and/or software component extracts the tilesfrom the least-cost path from source to sink. In step 2405, a module orother hardware and/or software component returns the extracted tiles.

According to an exemplary embodiment, there is provided a method fordetermining tiles for one or more given targets and candidate amplicons,comprising: (1) creating an overlap graph of candidate inserts, theoverlap graph comprising a set of vertices V and a set of edges E (e.g.,a graph G=(V,E)), such creating including (i) letting V equal the unionof the set of candidate amplicons (each of which being assigned acorresponding vertex) and the set consisting of a source element and asink element (e.g., V={amplicons}U{source,sink}), (ii) connecting thesource vertex to all initial vertices and the sink vertex to allterminal vertices, (iii) connecting each amplicon vertex to allsubsequent, proper, overlaps, and (iv) connecting rightmost vertices onthe left of a gap to leftmost vertices on the right of that gap; (2)defining an edge cost function or using a defined edge cost function;(3) finding a least cost path for such an edge cost function from thesource vertex to each vertex; (4) extracting the tiles from theleast-cost path from source to sink; and (5) returning the extractedtiles.

FIG. 25A illustrates a set of candidate amplicons for covering a giventarget region, each including an insert surrounded by a pair of primers,for tiling and pooling according to an exemplary embodiment. The dottedlines indicate the boundaries of the target region (on chromosome 19 inthis example).

FIG. 25B illustrates a set of vertices for generating a graph. Thevertices V includes 15 amplicon vertices (white) corresponding to the 15candidate amplicons illustrated in FIG. 25A together with a sourcevertex (light gray or green, left) and a sink vertex (dark grey or red,right). FIG. 26A illustrates the 15 candidate amplicons of FIG. 25A,except that three “initial” amplicons having at least some overlapbetween their insert and the beginning of the target region arehighlighted.

FIG. 26B illustrates the connection of a source vertex to three verticescorresponding to the initial amplicons of FIG. 26A with edges.

FIG. 27A illustrates the 15 candidate amplicons of FIG. 25A, except thatthree “terminal” amplicons having at least some overlap between theirinsert and the end of the target region are highlighted.

FIG. 27B illustrates the connection of a sink vertex to three verticescorresponding to the terminal amplicons of FIG. 27A with edges.

FIG. 28A illustrates the 15 candidate amplicons of FIG. 25A, except thatvarious amplicons for building internal edges are highlighted.

FIG. 28B illustrates the connection of some amplicon insert vertices tosubsequent, proper, overlaps according to an exemplary embodiment. Shownare arrows linking the 9767127 amplicon vertex to the 9767463 and9767519 amplicon vertices (whose inserts overlap the insert of the9767127 amplicon vertex) and arrows linking the 9767610 amplicon vertexto the 9767780 and 9767756 amplicon vertices (whose inserts overlap theinsert of the 9767610 amplicon vertex).

FIG. 29A illustrates the connection of additional amplicon insertvertices to subsequent, proper, overlaps according to an exemplaryembodiment. Also shown is a disconnect or gap that may arise if thecandidate amplicons do not fully cover the target.

FIG. 29B illustrates the 15 candidate amplicons of FIG. 25A, along withthe basis for the gap shown in FIG. 29A according to an exemplaryembodiment.

FIG. 30A illustrates three possible additional edges that could be usedfrom source to ink to tile the target in this example according to anexemplary embodiment. In an embodiment, of the possible paths the onewith a least cost may be selected.

FIG. 30B illustrates an exemplary definition of an edge cost functionassigning a cost to each one of the graph's edges linking ampliconvertices according to an exemplary embodiment.

FIG. 30C illustrates the least-cost path from source to ink in theexample of FIG. 30B according to an exemplary embodiment.

FIG. 31 illustrates the 15 candidate amplicons of FIG. 25A, except thatthe five amplicons corresponding to the vertices forming the least-costpath shown in FIG. 30C are highlighted.

According to an exemplary embodiment, the least-cost path may bedetermined using an O(|V|+|E|) algorithm. The least-cost path may bedetermined as follows: (1) for each vertex v, (i) initialize D[v] toinfinity (the least cost so far in the process) from the source vertexto v and initialize D[source] to zero, and (ii) initialize Pred[v] tonull (the predecessor of v in the least-cost path from the source vertexto v); and (2) for each vertex u in topological order, for each vertex vin adj[u], if D[u]+cost(u,v)<D[v], then let D[v]=D[u]+cost(u,v) andPred[v]=u. More information regarding algorithms for constructing pathson graphs may be found in Dijkstra, “A Note on Two Problems in Connexionwith Graphs,” Numerische Mathematik, vol. 1, 269-271 (1959), andSniedovich, “Dijkstra's algorithm revisited: the dynamic programmingconnexion,” Control and Cybernetics, vol. 35, 599-620 (2006), which areboth incorporated by reference herein in their entirety.

According to an exemplary embodiment, the cost of a path (e.g., ampliconplus “union” redundancy) may be the sum of the edges forming the path.In an embodiment, the cost of a path may be determined using Equation 1below:

$\begin{matrix}{{{cost}\; ({path})} = {\sum\limits_{{({u,v})} \in {path}}\; {{{cost}\left( {u,v} \right)}.}}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

According to an exemplary embodiment, the cost of an edge forming a pathmay be a sum of an amplicon cost and an overlap cost weighed using afactor α selected to blend the two costs, e.g., 0≦α≦1. In an embodiment,the cost of an edge forming the path may be determined using Equation 2below:

cost(u,ν)=αcost(ν.amplicon)+(1−α)cost(overlap(u,ν))  Equation 2

According to an exemplary embodiment, there is provided a method fordetermining a cost associated with a tiling or subset of amplicons. Inan embodiment, the cost of a tiling may be the sum of a cost of eachamplicon in the tiling plus a cost of any one or more insert overlaps.The cost of an amplicon may be assigned any suitable value in anysuitable predetermined range of values such that a low value imparts alow cost and a high value imparts a high cost so as to penalize variousundesirable characteristics such as, for example, off-targetamplification, primer-dimer propensity, etc. For example, the cost of anamplicon may be assigned a value from 1 to 10 such that a higher/lowercost is associated with a higher/lower level of undesirablecharacteristics (using any appropriate relationships, which may belinear/proportional or non-linear, for example). The cost of an insertoverlap may also similarly be assigned any suitable value in anysuitable predetermined range of values, for example, such that a lowvalue imparts a low cost and a high value imparts a high cost so as topenalize various undesirable characteristics such as, for example, theredundancy introduced by overlapping inserts. For example, the cost ofan insert overlap may also be assigned a value from 1 to 10. In anembodiment, one may opt to select a least-cost tiling from a set ofcandidate amplicons. In an embodiment, the cost of an insert overlap maybe determined based on the redundancy introduced by overlapping insertsusing Equation 3 below:

$\begin{matrix}{{{redundancy}\left( {{overlap}\left( {u,v} \right)} \right)} = \frac{{numBP}\left( {{overlap}\left( {u,v} \right)} \right)}{{numBP}\left( {{union}\left( {u,v} \right)} \right)}} & {{Equation}\mspace{14mu} 3}\end{matrix}$

According to an exemplary embodiment, the costs for amplicon andspecificity may be used by a tiling hardware or software component oftiler to calculate the cost of an edge. In an embodiment, a cost systemmay be used that allows a scaled cost calculation for a vertex in thetiler. The cost of an amplicon may be a composite of the amplicon costand the specificity cost. The amplicon cost may reflect the quality ofPCR amplification and the specificity cost may reflect the propensity ofthe amplicon to amplify off-target nucleotides.

In an embodiment, the cost of an amplicon may be determined as follows:Initially, a threshold may be used to filter primers that allowpotential off-target amplification. The filter may be based on a programsuch as e-PCR2 (NCBI), see Rotmistrovsky et al., “A web server forperforming electronic PCR,” Nucleic Acids Research, vol. 32, W108-W112(2004), and Schuler, “Sequence Mapping by Electronic PCR,” GenomeResearch, vol. 7, 541-550 (1997), which are both incorporated byreference herein in their entirety, or other similar programs capable ofbeing used for filtering primers. Various scoring strategies may be usedfor filtering. In various embodiments, the assays may be filtered thathave a sum of score of forward and reverse primer less than a certainminimal score threshold (e.g., 150) or show more than 50 hits with ascore less than some threshold (e.g., 200). Of course, numericalcut-offs may differ and will vary according to software used. In variousembodiments, scores used for filtering may be converted to a cost usinga conversion table such as illustrated below:

 0-19 170+ 10 20-39 150-169 9 40-59 130-149 8 60-79 110-129 7 80-99 90-109 6 100-119 70-89 5 120-149 50-69 4 150-179 30-49 3 180-210 10-292 210+ 0-9 1In this exemplary table, the first column represents sp_score, which isa specificity score; the second column represents sp_hits, which is thenumber of matches for the forward and reverse primers facing each other;and the third column is a corresponding cost. The first perfect match isfree, since it is the self match. In an embodiment, Acost is the costbased on binning the sp_score values, Bcost is the cost based on sp_hitsvalues, and the amplicon cost may be calculated using the formula:

AmpliconCost=0.9*Acost+0.1*Bcost.

FIG. 32 illustrates three amplicons assigned to a first pool and twoamplicons assigned to a second pool according to an exemplaryembodiment. The dotted lines indicate the boundaries of a target region(on chromosome 19 in this example). As illustrated, the amplicons in thefirst and second pools substantially cover the target regions, with theexception of the gaps.

According to an exemplary embodiment, there is provided a method forpooling amplicons into one or more pools of amplicons. In an embodiment,the method may pool the amplicons using one or more pooling criteria,which may related at least in part to pool sizes. In an embodiment, anumber of pools may be limited to a pre-determined maximum number ofpools, which may be 10 pools, for example, or which may be 50, 40, 30,20, 9, 8, 7, 6, 5, 4, 3, 2, or 1, for example, or any other positiveinteger. In an embodiment, a capacity of each pool (e.g, a maximal sizeof a pool) may also be limited to a pre-determined maximal value, whichmay be any fixed number of amplicons, and which may be about 10,000,about 7,500, about 5,000, about 2,500, about 2,000, about 1,500, about1,000, and about 500, or any value between those examples, for example,and which may be a maximum of 768 or 1,536 amplicons, for example. In anembodiment, a balance factor may be used, which may be a percentagebetween about 60% and 100%, or between about 65% and 95%, or betweenabout 70% and 90%, or about 90%, for example. In an embodiment, the sizeof a pool p may be constrained by the inequality set forth in Equation 4below:

$\begin{matrix}{{{size}(p)} \geq \left\lfloor {{balance}\; {Factor} \times {\max\limits_{q \in {pools}}\left\{ {{size}(q)} \right\}}} \right\rfloor} & {{Equation}\mspace{14mu} 4}\end{matrix}$

According to an exemplary embodiment, an intra pool distance may belimited by a minimal threshold distance, which may be, for example, aninteger number of base pairs (e.g., 50 base pairs, or 5, 10, 15, 20,etc., or any predetermined number of base pairs).

FIG. 33A illustrates a minimal distance between amplicons according toan exemplary embodiment. FIGS. 33B-D illustrate several problems,including primer “race condition” (see FIG. 33B), preferentialamplification of sub amplicons (see FIG. 33C), and super amplicons (seeFIG. 33D) that may be ameliorated by using a minimal distance asillustrated in FIG. 33A. In an embodiment, the distance may bedetermined using equation 5, for example. Such an approach may preventor reduces “race condition,” preferential amplification of subamplicons, and super amplicons.

dist(a,b)=max(a.start,b.start)−min(a.end,b.end)−1  Equation 5

According to an exemplary embodiment, amplicons may be disqualified frombeing added to a pool in certain circumstances. For example, an ampliconamp may not be added to a pool p if one or more of the followingcriteria are met: (1) the size of the pool p equals or exceeds the poolcapacity limit; (2) the size of the pool p equals the size of thelargest pool (e.g., size(p)=size(largest pool)) and the size of thesmallest pool is less than a floor (rounded down) value of the productof (i) the size of the pool p plus 1 and (ii) a balance factor (e.g.,size(smallest pool)<floor((size(p)+1) balanceFactor)); and (3) thedistance between amp and any other amplicon already in the pool is lessthan a minimum amplicon separation threshold (e.g., which distance maybe determined using Equation 5, for example).

FIG. 34 illustrates a method for pooling amplicons across a plurality ofpools according to an exemplary embodiment. In step 3401, a module orother hardware and/or software component sorts the amplicons bydecreasing priority. In step 3402, a module or other hardware and/orsoftware component starts a first available pool. In step 3403, a moduleor other hardware and/or software component empties all available pools.In step 3404, for each amplicon in the sorted amplicons, a module orother hardware and/or software component, (i) determines a list ofcompatible pools in which the amplicon could be multiplexed, (ii) ifthere is at least one compatible pool then places the amplicon in afirst compatible pool that maximizes the minimum distance between theamplicon and any other amplicon in the compatible pool, (iii) if thereis no compatible pool then if a new pool can be added adds a new pooland returns to step 3403, and (iv) if there is no compatible pool andnone can be added then puts the amplicon in an overflow (unpooled) listof amplicons. In step 3405, a module or other hardware and/or softwarecomponent outputs the pools.

According to an exemplary embodiment, a method for pooling ampliconsacross a plurality of pools comprises: (1) sorting the amplicons bydecreasing priority; (2) starting a first available pool; (3) emptyingall available pools; (4) for each amplicon in the sorted amplicons, (i)determining a list of compatible pools in which the amplicon could bemultiplexed, (ii) if there is at least one compatible pool then placingthe amplicon in a first compatible pool that maximizes the minimumdistance between the amplicon and any other amplicon in the compatiblepool, (iii) if there is no compatible pool then if a new pool can beadded add a new pool and return to step (3), and (iv) if there is nocompatible pool and none can be added then put the amplicon in anoverflow (unpooled) list of amplicons. The method may further compriseoutputting the amplicon pools.

Such a method may help ensure that the PCR pools cover as much of thetargets as do the candidate amplicons supplied as input. It may generatea small number of PCR pools (e.g., 2, 4, etc.). The primers in a poolwill not interact, which may help avoid undesired preferentialamplification, “race condition” competition for primer hybridization,and super-amplicons. Further, such a method may run very quickly andusing only a reasonable amount of memory.

FIG. 35 illustrates a method for tiling according to an exemplaryembodiment. In step 3501, a module or other hardware and/or softwarecomponent receives or provides as inputs a genomic target region and aset of candidate amplicons for the genomic target region. In step 3502,a module or other hardware and/or software component generates a graphcomprising a source vertex, a set of amplicon vertices arranged incorrespondence with the set of candidate amplicons, and a sink vertex.In step 3503, a module or other hardware and/or software componentdetermines a cost associated with one or more paths across the graphfrom the source vertex to the sink vertex via amplicon vertices. In step3504, a module or other hardware and/or software component extracts theamplicon vertices from the one of the one or more paths across the graphhaving a least cost associated therewith. In some embodiments, such amethod may be extended to a method for pooling amplicons across aplurality of pools by using as input the amplicons corresponding to theextracted vertices. For example, various steps such as described in FIG.34 may be performed on amplicons corresponding to the extracted verticesto pool the amplicons.

According to an exemplary embodiment, there is provided a methodcomprising: (1) receiving or providing as inputs a genomic target regionand a set of candidate amplicons for the genomic target region; (2)generating a graph comprising a source vertex, a set of ampliconvertices arranged in correspondence with the set of candidate amplicons,and a sink vertex; (3) determining a cost associated with one or morepaths across the graph from the source vertex to the sink vertex viaamplicon vertices; and (4) extracting the amplicon vertices from the oneof the one or more paths across the graph having a least cost associatedtherewith.

In various embodiments, the one or more paths may comprise a sequence ofamplicons wherein an ending portion of an insert of a first amplicon inthe sequence of amplicons overlaps a beginning portion of an insert of asecond amplicon in the sequence of amplicons. An ending portion of aninsert of the second amplicon in the sequence of amplicons may overlap abeginning portion of an insert of a third amplicon in the sequence ofamplicons. An ending portion of an insert of the third amplicon in thesequence of amplicons may overlap a beginning portion of an insert of afourth amplicon in the sequence of amplicons.

In various embodiments, the one or more paths may comprise a sequence ofN amplicons, N being a positive integer, wherein an ending portion of aninsert of an amplicon amp in the sequence of amplicons overlaps abeginning portion of an insert of an amplicon amp+1 in the sequence ofamplicons, wherein amp is an integer taking values 1, . . . , N−1. Theone or more paths may comprise a sequence of L=N+M amplicons, N and Mbeing positive integers, wherein an ending portion of an insert of anamplicon amp in the sequence of amplicons overlaps (which may includemerely touching) a beginning portion of an insert of an amplicon amp+1in the sequence of amplicons where amp is an integer taking values 1, .. . , N−1; wherein an ending portion of an insert of an amplicon amp inthe sequence of amplicons overlaps (which may include merely touching) abeginning portion of an insert of an amplicon amp+1 in the sequence ofamplicons where amp is an integer taking values N+1, . . . , N+M−1; andwherein there is a gap between an ending portion of an insert ofamplicon amp=N and a beginning portion of an insert of amplicon amp=N+1.

In various embodiments, the cost associated with each of the one or morepaths may be a sum of the cost of every edge of the path linking twoamplicon vertices. The cost associated with every edge of the pathlinking two amplicon vertices may be a sum of a first term related tothe cost of the edge's destination amplicon vertex and a second termrelated to the cost of an overlap between an insert of the edge'sdestination amplicon and an insert of the edge's origin amplicon. Thefirst term and the second term may be weighed by a blending factor suchthat the first term is multiplied by the blending factor or a functionthereof and the second term is multiplied by one minus the blendingfactor or a function thereof. The cost of an amplicon vertex may be anumerical value along a scale between a first value representing a lowerlevel of one or more undesirable characteristics selected from a groupcomprising at least a level of off-target amplification and a level ofprimer-dimer propensity and a second value representing a higher levelof the one or more undesirable characteristics. The cost of an overlapbetween an insert of the edge's destination amplicon and an insert ofthe edge's origin amplicon may be determined based on a redundancyintroduced by overlapping inserts. The cost of an overlap between aninsert of the edge's destination amplicon and an insert of the edge'sorigin amplicon may be a function of a quotient between a number of basepairs in an overlap between the insert of the edge's destinationamplicon and the insert of the edge's origin amplicon and a number ofbase pairs in a union of the insert of the edge's destination ampliconand the insert of the edge's origin amplicon.

In various embodiments, such a method may further comprise determining apooling of amplicons corresponding to the extracted amplicons into oneor more pools of amplicons. Determining a pooling of amplicons maycomprise limiting a number of amplicon pools and a capacity of eachamplicon pool. Determining a pooling of amplicons may comprise limitingthe number of amplicon pools to a threshold number between about 2 andabout 5 and limiting the capacity of each amplicon pool to a thresholdcapacity between about 500 amplicons and about 2,500 amplicons, forexample. Determining a pooling of amplicons may comprise limiting a sizeof any of the one or more pools based on a maximum size of other pools.

In various embodiments, determining a pooling of amplicons may compriselimiting an inclusion of one or more into a given pool based at least ona minimal threshold distance between amplicon sequences in the givenpool. The minimal threshold distance may be between about 5 base pairsand about 100 base pairs, for example.

According to an exemplary embodiment, there is provided a non-transitorymachine-readable storage medium comprising instructions which, whenexecuted by a processor, cause the processor to perform a methodcomprising: (1) receiving or providing as inputs a genomic target regionand a set of candidate amplicons for the genomic target region; (2)generating a graph comprising a source vertex, a set of ampliconvertices arranged in correspondence with the set of candidate amplicons,and a sink vertex; (3) determining a cost associated with one or morepaths across the graph from the source vertex to the sink vertex viaamplicon vertices; and (4) extracting the amplicon vertices from the oneof the one or more paths across the graph having a least cost associatedtherewith. In some embodiments, such a method may be extended to amethod for pooling amplicons across a plurality of pools by using asinput the amplicons corresponding to the extracted vertices. Forexample, various steps such as described in FIG. 34 may be performed onamplicons corresponding to the extracted vertices to pool the amplicons.

In various embodiments, such a non-transitory machine-readable storagemedium may comprise instructions which, when executed by a processor,cause the processor to perform a method further comprising determining apooling of amplicons corresponding to the extracted amplicons into oneor more pools of amplicons.

According to an exemplary embodiment, there is provided a system,comprising: (1) a machine-readable memory; and (2) a processorconfigured to execute machine-readable instructions, which, whenexecuted by the processor, cause the system to perform steps including:(a) receiving or providing as inputs a genomic target region and a setof candidate amplicons for the genomic target region; (b) generating agraph comprising a source vertex, a set of amplicon vertices arranged incorrespondence with the set of candidate amplicons, and a sink vertex;(c) determining a cost associated with one or more paths across thegraph from the source vertex to the sink vertex via amplicon vertices;and (d) extracting the amplicon vertices from the one of the one or morepaths across the graph having a least cost associated therewith. In someembodiments, such a system may be extended to a system for poolingamplicons across a plurality of pools by using as input the ampliconscorresponding to the extracted vertices. For example, various steps suchas described in FIG. 34 may be performed on amplicons corresponding tothe extracted vertices to pool the amplicons.

In various embodiments, the processor of such a system may further beconfigured to execute machine-readable instructions, which, whenexecuted by the processor, cause the system to perform steps includingdetermining a pooling of amplicons corresponding to the extractedamplicons into one or more pools of amplicons.

According to various exemplary embodiments, one or more features of anyone or more of the above-discussed teachings and/or exemplaryembodiments may be performed or implemented using appropriatelyconfigured and/or programmed hardware and/or software elements.Determining whether an embodiment is implemented using hardware and/orsoftware elements may be based on any number of factors, such as desiredcomputational rate, power levels, heat tolerances, processing cyclebudget, input data rates, output data rates, memory resources, data busspeeds, etc., and other design or performance constraints.

Examples of hardware elements may include processors, microprocessors,input(s) and/or output(s) (I/O) device(s) (or peripherals) that arecommunicatively coupled via a local interface circuit, circuit elements(e.g., transistors, resistors, capacitors, inductors, and so forth),integrated circuits, application specific integrated circuits, (ASIC),programmable logic devices (PLD), digital signal processors (DSP), fieldprogrammable gate array (FPGA), logic gates, registers, semiconductordevice, chips, microchips, chip sets, and so forth. The local interfacemay include, for example, one or more buses or other wired or wirelessconnections, controllers, buffers (caches), drivers, repeaters andreceivers, etc., to allow appropriate communications between hardwarecomponents. A processor is a hardware device for executing software,particularly software stored in memory. The processor can be any custommade or commercially available processor, a central processing unit(CPU), an auxiliary processor among several processors associated withthe computer, a semiconductor based microprocessor (e.g., in the form ofa microchip or chip set), a macroprocessor, or generally any device forexecuting software instructions. A processor can also represent adistributed processing architecture. The I/O devices can include inputdevices, for example, a keyboard, a mouse, a scanner, a microphone, atouch screen, an interface for various medical devices and/or laboratoryinstruments, a bar code reader, a stylus, a laser reader, aradio-frequency device reader, etc. Furthermore, the I/O devices alsocan include output devices, for example, a printer, a bar code printer,a display, etc. Finally, the I/O devices further can include devicesthat communicate as both inputs and outputs, for example, amodulator/demodulator (modem; for accessing another device, system, ornetwork), a radio frequency (RF) or other transceiver, a telephonicinterface, a bridge, a router, etc.

Examples of software may include software components, programs,applications, computer programs, application programs, system programs,machine programs, operating system software, middleware, firmware,software modules, routines, subroutines, functions, methods, procedures,software interfaces, application program interfaces (API), instructionsets, computing code, computer code, code segments, computer codesegments, words, values, symbols, or any combination thereof. A softwarein memory may include one or more separate programs, which may includeordered listings of executable instructions for implementing logicalfunctions. The software in memory may include a system for identifyingdata streams in accordance with the present teachings and any suitablecustom made or commercially available operating system (O/S), which maycontrol the execution of other computer programs such as the system, andprovides scheduling, input-output control, file and data management,memory management, communication control, etc.

According to various exemplary embodiments, one or more features of anyone or more of the above-discussed teachings and/or exemplaryembodiments may be performed or implemented using appropriatelyconfigured and/or programmed non-transitory machine-readable medium orarticle that may store an instruction or a set of instructions that, ifexecuted by a machine, may cause the machine to perform a method and/oroperations in accordance with the exemplary embodiments. Such a machinemay include, for example, any suitable processing platform, computingplatform, computing device, processing device, computing system,processing system, computer, processor, scientific or laboratoryinstrument, etc., and may be implemented using any suitable combinationof hardware and/or software. The machine-readable medium or article mayinclude, for example, any suitable type of memory unit, memory device,memory article, memory medium, storage device, storage article, storagemedium and/or storage unit, for example, memory, removable ornon-removable media, erasable or non-erasable media, writeable orre-writeable media, digital or analog media, hard disk, floppy disk,read-only memory compact disc (CD-ROM), recordable compact disc (CD-R),rewriteable compact disc (CD-RW), optical disk, magnetic media,magneto-optical media, removable memory cards or disks, various types ofDigital Versatile Disc (DVD), a tape, a cassette, etc., including anymedium suitable for use in a computer. Memory can include any one or acombination of volatile memory elements (e.g., random access, memory(RAM, such as DRAM, SRAM, SDRAM, etc.)) and nonvolatile memory elements(e.g., ROM, EPROM, EEROM, Flash memory, hard drive, tape, CDROM, etc.).Moreover, memory can incorporate electronic, magnetic, optical, and/orother types of storage media. Memory can have a distributed architecturewhere various components are situated remote from one another, but arestill accessed by the processor. The instructions may include anysuitable type of code, such as source code, compiled code, interpretedcode, executable code, static code, dynamic code, encrypted code, etc.,implemented using any suitable high-level, low-level, object-oriented,visual, compiled and/or interpreted programming language.

According to various exemplary embodiments, one or more features of anyone or more of the above-discussed teachings and/or exemplaryembodiments may be performed or implemented at least partly using adistributed, clustered, remote, or cloud computing resource.

According to various exemplary embodiments, one or more features of anyone or more of the above-discussed teachings and/or exemplaryembodiments may be performed or implemented using a source program,executable program (object code), script, or any other entity comprisinga set of instructions to be performed. When a source program, theprogram can be translated via a compiler, assembler, interpreter, etc.,which may or may not be included within the memory, so as to operateproperly in connection with the O/S. The instructions may be writtenusing (a) an object oriented programming language, which has classes ofdata and methods, or (b) a procedural programming language, which hasroutines, subroutines, and/or functions, which may include, for example,C, C++, Pascal, Basic, Fortran, Cobol, Perl, Java, and Ada.

According to various exemplary embodiments, one or more of theabove-discussed exemplary embodiments may include transmitting,displaying, storing, printing or outputting to a user interface device,a computer readable storage medium, a local computer system or a remotecomputer system, information related to any information, signal, data,and/or intermediate or final results that may have been generated,accessed, or used by such exemplary embodiments. Such transmitted,displayed, stored, printed or outputted information can take the form ofsearchable and/or filterable lists of runs and reports, pictures,tables, charts, graphs, spreadsheets, correlations, sequences, andcombinations thereof, for example.

Various additional exemplary embodiments may be derived by repeating,adding, or substituting any generically or specifically describedfeatures and/or components and/or substances and/or steps and/oroperating conditions set forth in one or more of the above-describedexemplary embodiments. Further, it should be understood that an order ofsteps or order for performing certain actions is immaterial so long asthe objective of the steps or action remains achievable, unlessspecifically stated otherwise. Furthermore, two or more steps or actionscan be conducted simultaneously so long as the objective of the steps oraction remains achievable, unless specifically stated otherwise.Moreover, any one or more feature, component, aspect, step, or othercharacteristic mentioned in one of the above-discussed exemplaryembodiments may be considered to be a potential optional feature,component, aspect, step, or other characteristic of any other of theabove-discussed exemplary embodiments so long as the objective of suchany other of the above-discussed exemplary embodiments remainsachievable, unless specifically stated otherwise.

Various additional exemplary embodiments may be derived by incorporatingin the above described exemplary embodiments one or more of the featuresdescribed in U.S. Pat. Appl. Publ. No. 2008/0228589 A1, published Sep.18, 2008, and U.S. Pat. Appl. Publ. No. 2004/0175733 A1, published Sep.9, 2004, the contents of both of which are incorporated by referenceherein in their entireties.

In some embodiments, the amplified target sequences generated by themethods disclosed herein represent at least 60%, 70%, 80%, 90%, or more,of one or more exons amplified from the plurality of target sequences.In one embodiment, amplified target sequences of the present inventionare about 90 to about 140 base pairs in length, about 100 to about 200base pairs in length, about 100 to about 300 base pairs in length, orabout 100 to about 400 base pairs in length. In one embodiment, theamplified target sequence includes the length of the forward primer andthe length of the complementary reverse primer for each primer pair. Inanother embodiment, the amplified target sequence length includes thelength of the reverse primer and the length of the complementary forwardprimer. In some embodiments, the length of the amplified target sequenceminus the forward and reverse primer lengths is about 40 base pairs toabout 350 base pairs. In some embodiments, the length of the amplifiedtarget sequences generated in the multiplex PCR reaction issubstantially the same. As defined herein, “substantially the same” withrespect to length of amplified target sequences generated via themethods disclosed herein refers to no more than 30% deviation innucleotide length across the total number of amplified target sequences.In one embodiment, the percent GC content of an amplicon is less than85%, less than 75%, less than 65%, less than 60%, or less than 50%. Inone embodiment, substantially all amplified target sequences within areaction contain between 30% and less than 85% GC content. In oneembodiment, where the nucleic acid molecules are obtained from anarchived or FFPE DNA sample, the length of the amplified target sequenceis typically about 100 to about 200 base pairs in length. In oneembodiment, if the nucleic acid sample is derived or obtained fromgenomic DNA, the length of the amplified target sequence can be about100 to about 500 base pairs in length.

In some embodiments, the amplified target sequences of the disclosedmethods can be used in various downstream analysis or assays with, orwithout, further purification or manipulation. For example, theamplified target sequences can be clonally amplified by techniques knownin the art, such a bridge amplification or emPCR to generate a templatelibrary that can be used in next generation sequencing. In someembodiments, the amplified target sequences of the disclosed methods orthe resulting template libraries can be used for single nucleotidepolymorphism (SNP) analysis, genotyping or epigenetic analysis, copynumber variation analysis, gene expression analysis, analysis of genemutations including but not limited to detection, prognosis and/ordiagnosis, detection and analysis of rare or low frequency allelemutations, nucleic acid sequencing including but not limited to de novosequencing, targeted resequencing and synthetic assembly analysis. Inone embodiment, amplified target sequences can be used to detectmutations at less than 5% allele frequency. In some embodiments, themethods disclosed herein can be used to detect mutations in a populationof nucleic acids at less than 4%, 3%, 2% or at about 1% allelefrequency. In another embodiment, amplified target sequences prepared asdescribed herein can be sequenced to detect and/or identify germline orsomatic mutations from a population of nucleic acid molecules.

In some embodiments, the forward and/or reverse target-specific primersin the target-specific primer pairs can be “complementary” or“substantially complementary” to the population of nucleic acidmolecules. As termed herein “substantially complementary to thepopulation of nucleic acid molecules” refers to percentagecomplementarity between the primer and the nucleic acid molecule towhich the primer will hybridize. Generally, the term “substantiallycomplementary” as used herein refers to at least 70% complementarity.Therefore, substantially complementary refers to a range ofcomplementarity of at least 70% but less than 100% complementaritybetween the primer and the nucleic acid molecule. A complementary primeris one that possesses 100% complementarity to the nucleic acid molecule.In one embodiment, each target-specific primer pair is designed tominimize cross-hybridization to another primer (or primer pair) in thesame multiple PCR reaction (i.e., reduce the prevalence ofprimer-dimers). In another embodiment, each target-specific primer pairis designed to minimize cross-hybridization to non-specific nucleic acidsequences in the population of nucleic acid molecules (i.e., minimizeoff-target hybridization). In one embodiment, each target-specificprimer is designed to minimize self-complementarity, formation ofhairpin structures or other secondary structures.

In some embodiments, the amplified target sequences are formed viapolymerase chain reaction. Extension of target-specific primers can beaccomplished using one or more DNA polymerases. In one embodiment, thepolymerase can be any Family A DNA polymerase (also known as pol Ifamily) or any Family B DNA polymerase. In some embodiments, the DNApolymerase can be a recombinant form capable of extendingtarget-specific primers with superior accuracy and yield as compared toa non-recombinant DNA polymerase. For example, the polymerase caninclude a high-fidelity polymerase or thermostable polymerase. In someembodiments, conditions for extension of target-specific primers caninclude ‘Hot Start’ conditions, for example Hot Start polymerases, suchas Amplitaq Gold® DNA polymerase (Applied Biosciences), Platinum® TaqDNA Polymerase High Fidelity (Invitrogen) or KOD Hot Start DNApolymerase (EMD Biosciences). Generally, a ‘Hot Start’ polymeraseincludes a thermostable polymerase and one or more antibodies thatinhibit DNA polymerase and 3′-5′ exonuclease activities at ambienttemperature. In some instances, ‘Hot Start’ conditions can include anaptamer.

In some embodiments, the polymerase can be an enzyme such as Taqpolymerase (from Thermus aquaticus), Tfi polymerase (from Thermusfiliformis), Bst polymerase (from Bacillus stearothermophilus), Pfupolymerase (from Pyrococcus furiosus), Tth polymerase (from Thermusthermophilus), Pow polymerase (from Pyrococcus woesei), Tli polymerase(from Thermococcus litoralis), Ultima polymerase (from Thermotogamaritima), KOD polymerase (from Thermococcus kodakaraensis), Pol I andII polymerases (from Pyrococcus abyssi) and Pab (from Pyrococcusabyssi). In some embodiments, the DNA polymerase can include at leastone polymerase such as Amplitaq Gold® DNA polymerase (AppliedBiosciences), Stoffel fragment of Amplitaq® DNA Polymerase (Roche), KODpolymerase (EMD Biosciences), KOD Hot Start polymerase (EMDBiosciences), Deep Vent™ DNA polymerase (New England Biolabs), Phusionpolymerase (New England Biolabs), Klentaq1 polymerase (DNA PolymeraseTechnology, Inc), Klentaq Long Accuracy polymerase (DNA PolymeraseTechnology, Inc), Omni KlenTaq™ DNA polymerase (DNA PolymeraseTechnology, Inc), Omni KlenTaq™ LA DNA polymerase (DNA PolymeraseTechnology, Inc), Platinum® Taq DNA Polymerase (Invitrogen), HemoKlentaq™ (New England Biolabs), Platinum® Taq DNA Polymerase HighFidelity (Invitrogen), Platinum® Pfx (Invitrogen), Accuprime™ Pfx(Invitrogen), or Accuprime™ Taq DNA Polymerase High Fidelity(Invitrogen).

In some embodiments, the DNA polymerase can be a thermostable DNApolymerase. In some embodiments, the mixture of dNTPs can be appliedconcurrently, or sequentially, in a random or defined order. In someembodiments, the amount of DNA polymerase present in the multiplexreaction is significantly higher than the amount of DNA polymerase usedin a corresponding single plex PCR reaction. As defined herein, the term“significantly higher” refers to an at least 3-fold greaterconcentration of DNA polymerase present in the multiplex PCR reaction ascompared to a corresponding single plex PCR reaction.

In some embodiments, the amplification reaction does not include acircularization of amplification product, for example as disclosed byrolling circle amplification.

In some embodiments, the methods of the disclosure include selectivelyamplifying target sequences in a sample containing a plurality ofnucleic acid molecules and ligating the amplified target sequences to atleast one Adaptersadapters and/or barcode. Adapters and barcodes for usein molecular biology library preparation techniques are well known tothose of skill in the art. The definitions of adapters and barcodes asused herein are consistent with the terms used in the art. For example,the use of barcodes allows for the detection and analysis of multiplesamples, sources, tissues or populations of nucleic acid molecules permultiplex reaction. A barcoded and amplified target sequence contains aunique nucleic acid sequence, typically a short 6-15 nucleotidesequence, that identifies and distinguishes one amplified nucleic acidmolecule from another amplified nucleic acid molecule, even when bothnucleic acid molecules minus the barcode contain the same nucleic acidsequence. The use of adapters allows for the amplification of eachamplified nucleic acid molecule in a uniformed manner and helps reducestrand bias. Adapters can include universal adapters or proprietyadapters both of which can be used downstream to perform one or moredistinct functions. For example, amplified target sequences prepared bythe methods disclosed herein can be ligated to an adapter that may beused downstream as a platform for clonal amplification. The adapter canfunction as a template strand for subsequent amplification using asecond set of primers and therefore allows universal amplification ofthe adapter-ligated amplified target sequence. In some embodiments,selective amplification of target nucleic acids to generate a pool ofamplicons can further comprise ligating one or more barcodes and/oradapters to an amplified target sequence. The ability to incorporatebarcodes enhances sample throughput and allows for analysis of multiplesamples or sources of material concurrently. In one example, amplifiedtarget nucleic acid molecules prepared by the disclosed methods can beligated to Ion Torrent™ Sequencing AdaptersAdapters (A and P1Adaptersadapters, sold as a component of the Ion Fragment Library Kit,Life Technologies, Part No. 4466464) or Ion Torrent™ DNA Barcodes (LifeTechnologies, Part No. 4468654).

The methods disclosed herein are directed to the amplification ofmultiple target sequences via polymerase chain reaction (PCR). In someembodiments the multiplex PCR comprises hybridizing one or moretarget-specific primer pairs to a nucleic acid molecule, extending theprimers of the target-specific primer pairs via template dependentsynthesis in the presence of a DNA polymerase and dNTPs; repeating thehybridization and extension steps for sufficient time and sufficienttemperature there generating a plurality of amplified target sequences.In some embodiments, the steps of the multiplex amplification reactionmethod can be performed in any order.

The amount of nucleic acid material required for successful multiplexamplification can be about 1 ng. In some embodiments, the amount ofnucleic acid material can be about 10 ng to about 50 ng, about 10 ng toabout 100 ng, or about 1 ng to about 200 ng of nucleic acid material.Higher amounts of input material can be used, however one aspect of thedisclosure is to selectively amplify a plurality of target sequence froma low (ng) about of starting material.

The multiplex PCR amplification reactions disclosed herein can include aplurality of “cycles” typically performed on a thermocycler. Generally,each cycle includes at least one annealing step and at least oneextension step. In one embodiment, a multiplex PCR amplificationreaction is performed wherein target-specific primer pairs arehybridized to a target sequence; the hybridized primers are extendedgenerating an extended primer product/nucleic acid duplex; the extendedprimer product/nucleic acid duplex is denatured allowing thecomplementary primer to hybridize to the extended primer product,wherein the complementary primer is extended to generate a plurality ofamplified target sequences. In one embodiment, the methods disclosedherein have about 5 to about 18 cycles per preamplification reaction.The annealing temperature and/or annealing duration per cycle can beidentical; can include incremental increases or decreases, or acombination of both. The extension temperature and/or extension durationper cycle can be identical; can include incremental increases ordecreases, or a combination of both. For example, the annealingtemperature or extension temperature can remain constant per cycle. Insome embodiments, the annealing temperature can remain constant eachcycle and the extension duration can incrementally increase per cycle.In some embodiments, increases or decreases in duration can occur in 15second, 30 second, 1 minute, 2 minute or 4 minute increments. In someembodiments, increases or decrease in temperature can occur as 0.5, 1,2, 3, or 4 Celsius deviations. In some embodiments, the amplificationreaction can beconducted using hot-start PCR techniques. Thesetechniques include the use of a heating step (>60° C.) beforepolymerization begins to reduce the formation of undesired PCR products.Other techniques such as the reversible inactivation or physicalseparation of one or more critical reagents of the reaction, for examplethe magnesium or DNA polymerase can be sequestered in a wax bead, whichmelts as the reaction is heated during the denaturation step, releasingthe reagent only at higher temperatures. The DNA polymerase can also bekept in an active state by binding to an aptamer or an antibody. Thisbinding is disrupted at higher temperatures, releasing the functionalDNA polymerase that can proceed with the PCR unhindered.

In some embodiments, the disclosed methods can optionally includedestroying one or more primer-containing amplification artifacts, e.g.,primer-dimers, dimer-dimers or superamplicons. In some embodiments, thedestroying can optionally include treating the primer and/oramplification product so as to cleave specific cleavable groups presentin the primer and/or amplification product. In some embodiments, thetreating can include partial or complete digestion of one or moretarget-specific primers. In one embodiment, the treating can includeremoving at least 40% of the target specific primer from theamplification product. The cleavble treatment can include enzymatic,acid, alkali, thermal, photo or chemical activity. The cleavabletreatment can result in the cleavage or other destruction of thelinkages between one or more nucleotides of the primer, or between oneor more nucleotides of the amplification product. The primer and/or theamplification product can optionally include one or more modifiednucleotides or nucleobases. In some embodiments, the cleavage canselectively occur at these sites, or adjacent to the modifiednucleotides or nucleobases. In some embodiments, the cleavage ortreatment of the amplified target sequence can result in the formationof a phosphorylated amplified target sequence. In some embodiments, theamplified target sequence is phosphorylated at the 5′ terminus.

In some embodiments, the template, primer and/or amplification productincludes nucleotides or nucleobases that can be recognized by specificenzymes. In some embodiments, the nucleotides or nucleobases can bebound by specific enzymes. Optionally, the specific enzymes can alsocleave the template, primer and/or amplification product at one or moresites. In some embodiments, such cleavage can occur at specificnucleotides within the template, primer and/or amplification product.For example, the template, primer and/or amplification product caninclude one or more nucleotides or nucleobases including uracil, whichcan be recognized and/or cleaved by enzymes such as uracil DNAglycosylase (UDG, also referred to as UNG) or formamidopyrimidine DNAglycosylase (Fpg). The template, primer and/or amplification product caninclude one or more nucleotides or nucleobases including RNA-specificbases, which can be recognized and/or cleaved by enzymes such as RNAseH.In some embodiments, the template, primer and/or amplification productcan include one or more abasic sites, which can be recognized and/orcleaved using various proofreading polymerases or apyrase treatments. Insome embodiments, the template, primer and/or amplification product caninclude 7,8-dihydro-8-oxoguanine (8-oxoG) nucleobases, which can berecognized or cleaved by enzymes such as Fpg. In some embodiments, oneor more amplified target sequences can be partially digested by a FuPareagent.

In some embodiments, the primer and/or amplification product includesone or more modified nucleotides including bases that bind, e.g., basepair, with other nucleotides, for example nucleotides in a complementarynucleic acid strand, via chemical linkages. In some embodiments, thechemical linkages are subject to specific chemical attack thatselectively cleaves the modified nucleotides (or selectively cleaves oneor more covalent linkages between the modified nucleotides and adjacentnucleotides within the primer and/or amplification product) but leavesthe other nucleotides unaffected. For example, in some embodimentsmodified nucleotides can form disulfite linkages with other nucleotidesin a complementary strand. Such disulfite linkages can be oxidized viasuitable treatments. Similarly, certain modified nucleotides can basepair with other nucleotides in a complementary nucleic acid strandthrough linkages that can be selectively disrupted via alkali treatment.In some embodiments, the primer and/or amplification product includesone or more modified nucleotides that bind, e.g., base pair, with othernucleotides in a complementary nucleic acid strand through linkagesexhibiting decreased thermal stability relative to typical base pairinglinkages formed between natural bases. Such reduced-thermal stabilitylinkages can be selectively disrupted through exposure of the primerand/or amplification product to elevated temperatures followingamplification.

An exemplary embodiment is depicted in FIG. 1, which depicts a schematicof degradable amplification primers. The amplification primers arebisulfite in design, with either a 5′ universal forward amplificationsequence linked to a 3′ target-specific forward primer, or a 5′universal reverse amplification sequence linked to a 3′ target-specificreverse primer. Both primers contain modified nucleotides.

In some embodiments, primers are synthesized that are complementary to,and can hybridize with, discrete segments of a nucleic acid templatestrand, including: a primer that can hybridize to the 5′ region of thetemplate, which encompasses a sequence that is complementary to eitherthe forward or reverse amplification primer. In some embodiments, theforward primers, reverse primers, or both, share no common nucleic acidsequence, such that they hybridize to distinct nucleic acid sequences.For example, target-specific forward and reverse primers can be preparedthat do not compete with other primer pairs within the primer pool toamplify the same nucleic acid sequence. In this example, primer pairsthat do not compete with other primer pairs in the primer pool assist inthe reduction of non-specific or spurious amplification products. Insome embodiments, the forward and reverse primers of each primer pairare unique, in that the nucleotide sequence for each primer isnon-complementary and non-identical to the other primer in the primerpair. In some embodiments, the primer pair can differ by at least 10%,at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 75%, at least 80%, at least 85%, or at least 90%nucleotide identity. In some embodiments, the forward and reverseprimers in each primer pair are non-complementary or non-identical toother primer pairs in the primer pool or multiplex reaction. Forexample, the primer pairs within a primer pool or multiplex reaction candiffer by at least 5%, at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, or at least 70% nucleotideidentity to other primer pairs within the primer pool or multiplexreaction. Generally, primers are designed to minimize the formation ofprimer-dimers, dimer-dimers or other non-specific amplificationproducts. Typically, primers are optimized to reduce GC bias and lowmelting temperatures (T_(m)) during the amplification reaction. In someembodiments, the primers are designed to possess a T_(m) of about 55° C.to about 72° C. In some embodiments, the primers of a primer pool canpossess a T_(m) of about 59° C. to about 70° C., 60° C. to about 68° C.,or 60° C. to about 65° C. In some embodiments, the primer pool canpossess a T_(m) that does not deviate by more than 5° C.

In some embodiments, the target-specific primers do not contain acarbon-spacer or terminal linker. In some embodiments, thetarget-specific primers or amplified target sequences do not contain anenzymatic, magnetic, optical or fluorescent label.

The template can include a 3′ region that contains the sequence foreither the upstream or downstream regions surrounding a particular geneor region of interest, such that the region of interest is bracketed bya forward amplification/upstream gene-specific fusion, and a reverseamplification/downstream region of interest fusion primer. In someembodiments, an internal separator sequence can separate the templateregions that can hybridize to the amplification and gene-specificprimers, and this may act as a key or barcode for subsequent downstreamapplications such as sequencing, etc. In some embodiments, a barcode orkey can be incorporated into each of the amplification products toassist with data analysis and for example, cataloging. In someembodiments, the barcodes can be Ion Torrent™ DNA barcodes (LifeTechnologies).

In some embodiments, the primer includes a sufficient number of modifiednucleotides to allow functionally complete degradation of the primer bythe cleavage treatment, but not so many as to interfere with theprimer's specificity or functionality prior to such cleavage treatment,for example in the amplification reaction. In some embodiments, theprimer includes at least one modified nucleotide, but no greater than75% of nucleotides of the primer are modified.

In some embodiments, multiple different primers including at least onemodified nucleotide can be used in a single amplification reaction. Forexample, multiplexed primers including modified nucleotides can be addedto the amplification reaction mixture, where each primer (or set ofprimers) selectively hybridizes to, and promotes amplification ofdifferent target nucleic acid molecules within the nucleic acidpopulation. In some embodiments, different primer combinations can beadded to the amplification reaction at plexy of at least about 24, 96,384, 768, 1000, 2000, 3000, 6000 or 10000, or more (where “plexy”indicates the total number of different targets that can theoreticallybe amplified in a sequence-specific manner in the amplificationreaction). In some embodiments, the modified primers contain at leastone modified nucleotide near or at the termini of the primer. In someembodiments, the modified primers contain two or more modifiednucleotides within the primer sequence. In an exemplary embodiment, theprimer sequence contains a uracil near, or at, the termini of the primersequence. For the purposes of this disclosure “near” or “at the termini”of the primer sequences refers up to 10 nucleotides from the termini ofthe primer sequence. In some embodiments, the primer sequence contains auracil located at, or about, the center nucleotide position of theprimer sequence. For the purposes of this disclosure “at, or about thecenter nucleotide position of the primer sequence” refers to theincorporation of a uracil moiety at the center nucleotide of the primersequence or within eight nucleotides, in either a 3′ or 5′ directionflanking the center nucleotide. In one embodiment, the target-specificprimer sequence can contain a modified nucleobase at or about the centernucleotide position and contain a modified nucleobase at the 3′ and/or5′ terminus. In some embodiments, the length of the forward or reverseprimer sequence can be about 15 to about 40 bases in length. In someembodiments, the T_(m) of the primer sequence used in the multiplexreaction can be about 55° C. to about 72° C. In some embodiments, theprimer pairs are designed to amplify sequences from the target nucleicacid molecules or amplicons that are about 100 base pairs to about 500base pairs in length.

In some embodiments, the amplification reactions are conducted inparallel within a single reaction phase (for example, within the sameamplification reaction mixture within a single tube). In some instances,an amplification reaction can generate a mixture of products includingboth the intended amplicon product as well as unintended, unwanted,nonspecific amplification artifacts such as primer-dimers. Postamplification, the reactions are then treated with any suitable agentthat will selectively cleave or otherwise selectively destroy thenucleotide linkages of the modified nucleotides within the excessunincorporated primers and the amplification artifacts without cleavingor destroying the specification amplification products. For example, theprimers can include uracil-containing nucleobases that can beselectively cleaved using UNG/UDG (optionally with heat and/or alkali).In some embodiments, the primers can include uracil-containingnucleotides that can be selectively cleaved using UNG and Fpg. In someembodiments, the cleavage treatment includes exposure to oxidizingconditions for selective cleavage of dithiols, treatment with RNAseH forselective cleavage of modified nucleotides including RNA-specificmoieties (e.g., ribose sugars, etc.), and the like. This cleavagetreatment can effectively fragment the original amplification primersand non-specific amplification products into small nucleic acidfragments that include relatively few nucleotides each. Such fragmentsare typically incapable of promoting further amplification at elevatedtemperatures. Such fragments can also be removed relatively easily fromthe reaction pool through the various post-amplification cleanupprocedures known in the art (e.g., spin columns, NaEtOH precipitation,etc).

In some embodiments, amplification products following cleavage or otherselective destruction of the nucleotide linkages of the modifiednucleotides are optionally treated to generate amplification productsthat possess a phosphate at the 5′ termini. In some embodiments, thephosphorylation treatment includes enzymatic manipulation to produce 5′phosphorylated amplification products. In one embodiment, enzymes suchas polymerases can be used to generate 5′ phosphorylated amplificationproducts. For example, T4 polymerase can be used to prepare 5′phosphorylated amplicon products. Klenow can be used in conjunction withone or more other enzymes to produce amplification products with a 5′phosphate. In some embodiments, other enzymes known in the art can beused to prepare amplification products with a 5′ phosphate group. Forexample, incubation of uracil nucleotide containing amplificationproducts with the enzyme UDG, Fpg and T4 polymerase can be used togenerate amplification products with a phosphate at the 5′ termini. Itwill be apparent to one of skill in the art that other techniques, otherthan those specifically described herein, can be applied to generatephosphorylated amplicons. It is understood that such variations andmodifications that are applied to practice the methods, systems, kits,compositions and apparatuses disclosed herein, without resorting toundue experimentation are considered within the scope of the disclosure.

In some embodiments, primers that are incorporated in the intended(specific) amplification products, these primers are similarly cleavedor destroyed, resulting in the formation of “sticky ends” (e.g., 5′ or3′ overhangs) within the specific amplification products. Such “stickyends” can be addressed in several ways. For example, if the specificamplification products are to be cloned, the overhang regions can bedesigned to complement overhangs introduced into the cloning vector,thereby enabling sticky ended ligations that are more rapid andefficient than blunt ended ligations. Alternatively, the overhangs mayneed to be repaired (as with several next-generation sequencingmethods). Such repair can be accomplished either through secondaryamplification reactions using only forward and reverse amplificationprimers (in the embodiment shown in FIG. 1, this corresponds to A and P1primers) comprised of only natural bases. In this manner, subsequentrounds of amplification rebuild the double-stranded templates, withnascent copies of the amplicon possessing the complete sequence of theoriginal strands prior to primer destruction. Alternatively, the stickyends can be removed using some forms of fill-in and ligation processing,wherein the forward and reverse primers are annealed to the templates. Apolymerase can then be employed to extend the primers, and then aligase, optionally a thermostable ligase, can be utilized to connect theresulting nucleic acid strands. This could obviously be alsoaccomplished through various other reaction pathways, such as cyclicalextend-ligation, etc. In some embodiments, the ligation step can beperformed using one or more DNA ligases.

The amplification reaction can include any reaction that increases thecopy number of a nucleic acid molecule, optionally in a cyclicalfashion, and can include without limitation isothermal amplification(for example, rolling circle amplification or isothermal amplificationas described in U.S. Provisional Application Nos. 61/424,599, 61/445,324and 61/451,919, hereby incorporated by reference in their entireties),amplification using thermocycling, and the like.

In some embodiments, the disclosure generally relates to methods forsingle-tube multiplex PCR. In some embodiments, the method forsingle-tube multiplex PCR can include target-specific or exon-specificprimers. In some embodiments, the exon-specific or target-specificprimers can include at least one uracil nucleotide. In some embodiments,single-tube multiplex PCR can include selective amplification of atleast 1000, 2000, 3000, 4000, 5000, 6000 or more target nucleic acidmolecules using target-specific or exon-specific uracil based primers.

In some embodiments, the disclosure relates generally to methods forgenerating a target-specific or exon-specific amplicon library. In someembodiments, the amplicon library generated using target-specific orexon-specific primers can be associated with mutations of human cancers.In some embodiments, the mutations can be in the KRAS, BRAF and/or EGFRgenes. In some embodiments, the amplicon library can be generated fromgenomic DNA or formalin-fixed, paraffin-embedded (FFPE) tissue. In someembodiments, the amplicons of the amplicon library prepared using themethods disclosed herein can be about 100 to about 300 base pairs inlength, about 100 to about 250 base pairs in length, about 120 to about220 base pairs in length or about 135 to about 205 base pairs in length.In some embodiments, the amplicon library can be prepared using primerpairs that are targeted to cancer specific mutations. In someembodiments, the primer pairs can be directed to non-cancer relatedmutations, such as inherited diseases, e.g., cystic fibrosis and thelike. In some embodiments, the primer pairs can be used to generateamplicons that once sequenced by any sequencing platform, includingsemi-conductor sequencing technology can be used to detect geneticmutations such as inversion, deletions, point mutations and variationsin copy number.

In some embodiments, the primer pairs used to produce an ampliconlibrary can result in the amplification of target-specific nucleic acidmolecules possessing one or more of the following metrics: greater than97% target coverage at 20× if normalized to 100× average coverage depth;greater than 97% of bases with greater than 0.2× mean; greater than 90%base without strand bias; greater than 95% of all reads on target;greater than 99% of bases with greater than 0.01× mean; and greater than99.5% per base accuracy.

In some embodiments, the amplicon library can be used to detect and/oridentify known mutations or de novo mutations in a sample.

In some embodiments, the amplicon library prepared using target-specificprimer pairs can be used in downstream enrichment applications such asemulsion PCR or bridge PCR. In some embodiments, the amplicon librarycan be used in an enrichment application and a sequencing application.For example, an amplicon library can be sequenced using any suitable DNAsequencing platform. In some embodiments, an amplicon library can besequenced using an Ion Torrent PGM Sequencer (Life Technologies). Insome embodiments, a PGM sequencer can be coupled to server that appliesparameters or software to determine the sequence of the amplified targetnucleic acid molecules. In some embodiments, the amplicon library can beprepared, enriched and sequenced in less than 24 hours. In someembodiments, the amplicon library can be prepared, enriched andsequenced in approximately 9 hours. In some embodiments, an ampliconlibrary can be a paired library, that is, a library that containsamplicons from a tumor sample and amplicons from a non-diseased sample.Each pair can be aligned, to detect and/or identify mutations present inthe target nucleic acid molecules.

In some embodiments, methods for generating an amplicon library caninclude: amplifying genomic DNA targets using exon-specific ortarget-specific primers to generate amplicons; purifying the ampliconsfrom the input DNA and primers; phosphorylating the amplicons; ligatingAdaptersadapters to the phosphorylated amplicons; purifying the ligatedamplicons; nick-translating the amplified amplicons; and purifying thenick-translated amplicons to generate the amplicon library. In someembodiments, additional amplicon library manipulations can be conductedfollowing the step of amplification of genomic DNA targets to generatethe amplicons. In some embodiments, any combination of additionalreactions can be conducted in any order, and can include: purifying;phosphorylating; ligating Adaptersadapters; nick-translating;amplification and/or sequencing. In some embodiments, any of thesereactions can be omitted or can be repeated. It will be readily apparentto one of skill in the art that the method can repeat or omit any one ormore of the above steps. It will also be apparent to one of skill in theart that the order and combination of steps may be modified to generatethe required amplicon library, and is not therefore limited to theexemplary methods provided.

A phosphorylated amplicon can be joined to an adapter to conduct a nicktranslation reaction, subsequent downstream amplification (e.g.,template preparation), or for attachment to particles (e.g., beads), orboth. For example, an adapter that is joined to a phosphorylatedamplicon can anneal to an oligonucleotide capture primer which isattached to a particle, and a primer extension reaction can be conductedto generate a complementary copy of the amplicon attached to theparticle or surface, thereby attaching an amplicon to a surface orparticle. AdaptersAdapters can have one or more amplification primerhybridization sites, sequencing primer hybridization sites, barcodesequences, and combinations thereof. In some embodiments, ampliconsprepared by the methods disclosed herein can be joined to one or moreIon Torrent™ compatible Adaptersadapters to construct an ampliconlibrary. Amplicons generated by such methods can be joined to one ormore Adaptersadapters for library construction to be compatible with anext generation sequencing platform. For example, the amplicons producedby the teachings of the present disclosure can be attached toAdaptersadapters provided in the Ion Fragment Library Kit (LifeTechnologies, Catalog No. 4466464).

In some embodiments, amplification of genomic DNA targets (such as highmolecular weight DNA) or FFPE samples can be conducted using a 2×AmpliSeq Hi Fi Master Mix. In some embodiments, the AmpliSeq Hi FiMaster Mix can include glycerol, dNTPs, and a DNA polymerase, such asPlatinum® Taq DNA polymerase High Fidelity. In some embodiments, the 2×AmpliSeq Hi Fi Master Mix can further include at least one of thefollowing: a preservative, magnesium sulfate, tris-sulfate and/orammonium sulfate.

In some embodiments, amplification of genomic DNA targets (such as highmolecular weight DNA) or FFPE samples can be conducted using a 5× IonAmpliSeq Hi Fi Master Mix. In some embodiments, the 5× Ion AmpliSeq HiFi Master Mix can include glycerol, dNTPs, and a DNA polymerase such asPlatinum® Taq DNA polymerase High Fidelity. In some embodiments, the 5×Ion AmpliSeq Hi Fi Master Mix can further include at least one of thefollowing: a preservative, magnesium chloride, magnesium sulfate,tris-sulfate and/or ammonium sulfate.

In some embodiments, phosphorylation of the amplicons can be conductedusing a FuP reagent. In some embodiments, the FuP reagent can include aDNA polymerase, a DNA ligase, at least one uracil cleaving or modifyingenzyme, and/or a storage buffer. In some embodiments, the FuP reagentcan further include at least one of the following: a preservative and/ora detergent.

In some embodiments, phosphorylation of the amplicons can be conductedusing a FuPa reagent. In some embodiments, the FuPa reagent can includea DNA polymerase, at least one uracil cleaving or modifying enzyme, anantibody and/or a storage buffer. In some embodiments, the FuPa reagentcan further include at least one of the following: a preservative and/ora detergent. In some embodiments, the antibody is provided to inhibitthe DNA polymerase and 3′-5′ exonuclease activities at ambienttemperature.

In some embodiments, the amplicon library produced by the teachings ofthe present disclosure are sufficient in yield to be used in a varietyof downstream applications including the Ion Xpress™ Template Kit usingan Ion Torrent™ PGM system (e.g., PCR-mediated addition of the nucleicacid fragment library onto Ion Sphere™ Particles) (Life Technologies,Part No. 4467389). For example, instructions to prepare a templatelibrary from the amplicon library can be found in the Ion XpressTemplate Kit User Guide (Life Technologies, Part No. 4465884), herebyincorporated by reference in its entirety. Instructions for loading thesubsequent template library onto the Ion Torrent™ Chip for nucleic acidsequencing are described in the Ion Sequencing User Guide (Part No.4467391), hereby incorporated by reference in its entirety. In someembodiments, the amplicon library produced by the teachings of thepresent disclosure can be used in paired end sequencing (e.g.,paired-end sequencing on the Ion Torrent™ PGM system (Life Technologies,Part No. MAN0006191), hereby incorporated by reference in its entirety.

It will be apparent to one of ordinary skill in the art that numerousother techniques, platforms or methods for clonal amplification such aswildfire PCR and bridge amplification can be used in conjunction withthe amplified target sequences of the present disclosure. It is alsoenvisaged that one of ordinary skill in art upon further refinement oroptimization of the conditions provided herein can proceed directly tonucleic acid sequencing (for example using the Ion Torrent PGM™ orProton™ sequencers, Life Technologies) without performing a clonalamplification step.

In some embodiments, at least one of the amplified targets sequences tobe clonally amplified can be attached to a support or particle. Thesupport can be comprised of any suitable material and have any suitableshape, including, for example, planar, spheroid or particulate. In someembodiments, the support is a scaffolded polymer particle as describedin U.S. Published App. No. 20100304982, hereby incorporated by referencein its entirety.

In some embodiments, nucleic acid sequencing of the amplified targetsequences produced by the teachings of this disclosure include de novosequencing or targeted resequencing. In some embodiments, nucleic acidsequencing further includes comparing the nucleic acid sequencingresults of the amplified target sequences against a reference nucleicacid sample. In some embodiments, the reference sample can be normaltissue or well documented tumor sample. In some embodiments, nucleicacid sequencing of the amplified target sequences further includesdetermining the presence or absence of a mutation within the nucleicacid sequence. In some embodiments, the method further includescorrelating the presence of a mutation with drug susceptibly, prognosisof treatment and/or organ rejection. In some embodiments, nucleic acidsequencing includes the identification of genetic markers associatedwith cancer and/or inherited diseases. In some embodiments, nucleic acidsequencing includes the identification of copy number variation in asample under investigation.

In some embodiments, a kit is provided for amplifying multiple targetsequences from a population of nucleic acid molecules in a singlereaction. In some embodiments, the kit includes a plurality oftarget-specific primer pairs containing one or more cleavable groups,one or more DNA polymerases, a mixture of dNTPs and at least onecleaving reagent. In one embodiment, the cleavable group can be8-oxo-deoxyguanosine, deoxyuridine or bromodeoxyuridine. In someembodiments, the at least one cleaving reagent includes RNaseH, uracilDNA glycosylase, Fpg or alkali. In one embodiment, the cleaving reagentcan be uracil DNA glycosylase. In some embodiments, the kit is providedto perform multiplex PCR in a single reaction chamber or vessel. In someembodiments, the kit includes at least one DNA polymerase, which can bea thermostable DNA polymerase. In some embodiments, the concentration ofthe one or more DNA polymerases is present in a 3-fold excess ascompared to a single PCR reaction. In some embodiments, the finalconcentration of each target-specific primer pair is present at about 25nM to about 50 nM. In one embodiment, the final concentration of eachtarget-specific primer pair can be present at a concentration that is50% lower than conventional single plex PCR reactions. In someembodiments, the kit provides amplification of at least 100, 500, 1000,3000, 6000, 10000, 12000, or more, target sequences from a population ofnucleic acid molecules in a single reaction chamber.

In some embodiments, the kit further comprises one or more adapters,barcodes, and/or antibodies.

The following description of various exemplary embodiments is exemplaryand explanatory only and is not to be construed as limiting orrestrictive in any way. Other embodiments, features, objects, andadvantages of the present teachings will be apparent from thedescription and accompanying drawings, and from the claims.

Although the present description described in detail certain exemplaryembodiments, other embodiments are also possible and within the scope ofthe present invention. Variations and modifications will be apparent tothose skilled in the art from consideration of the specification andfigures and practice of the teachings described in the specification andfigures, and the claims.

EXAMPLES Example 1 Library Preparation

PCR Amplify Genomic DNA Targets

A multiplex polymerase chain reaction was performed to amplify 384individual amplicons across a genomic DNA sample. A pool of greater than32,000 primers was originally developed covering more than 300 genes. Arepresentative list of genes that were incorporated for investigationwhile synthesizing the initial primer pool is provided in Table 1. Asub-set of the primer pool known as “HSMv12” was used to generate thedata presented in FIG. 5 (primers from HSMv12 are presented in Table 2).Each primer pair in the primer pool was designed to contain at least oneuracil nucleotide near the terminus of each forward and reverse primer.Each primer pair was also designed to selectively hybridize to, andpromote amplification of a specific gene or gene fragment of the genomicDNA sample.

To a single well of a 96-well PCR plate was added 5 microliters ofHSMv12 Primer Pool containing 384 primer pairs at a concentration of 15μM in TE, 10-50 ng genomic DNA and 10 microliters of an amplificationreaction mixture (2× AmpliSeq HiFi Master Mix) that can includeglycerol, dNTPs, and Platinum® Taq High Fidelity DNA Polymerase(Invitrogen, Catalog No. 11304) to a final volume of 20 microliters withDNase/RNase Free Water (Life Technologies, CA, Part No. 600004).

The PCR plate was sealed and loaded into a thermal cycler (GeneAmp® PCRsystem 9700 Dual 96-well thermal cycler (Life Technologies, CA, Part No.N8050200 and 4314445)) and run on the following temperate profile togenerate the preamplified amplicon library.

An initial holding stage was performed at 98° C. for 2 minutes, followedby 16 cycles of denaturing at 98° C. for 15 seconds and an annealing andextending stage at 60° C. for 4 minutes. After cycling, the preamplifiedamplicon library was held at 4° C. until proceeding to the purificationstep outlined below. A schematic of an exemplary library amplificationprocess is shown in FIG. 2.

Purify the Amplicons from Input DNA and Primers

Two rounds of Agencourt® AMPure® XP Reagent (Beckman Coulter, CA)binding, wash, and elution at 0.6× and 1.2× volume ratios were found toremove genomic DNA and unbound or excess primers. The amplification andpurification step outlined herein produces amplicons of about 100 bp toabout 600 bp in length.

In a 1.5 ml LoBind tube (Eppendorf, Part No. 022431021), thepreamplified amplicon library (20 microliters) was combined with 12microliters (0.6× volumes) of Agencourt® AMPure® XP reagent (BeckmanCoulter, CA). The bead suspension was pipetted up and down to thoroughlymix the bead suspension with the preamplified amplicon library. Thesample was then pulse-spin and incubated for 5 minutes at roomtemperature.

The tube containing the sample was placed on a magnetic rack such as aDynaMag™-2 spin magnet (Life Technologies, CA, Part No. 123-21D) for 2minutes to capture the beads. Once the solution cleared, the supernatantwas transferred to a new tube, where 24 microliters (1.2× volume) ofAgenCourt® AMPure® XP beads (Beckman Coulter, CA) were added to thesupernatant. The mixture was pipetted to ensure the bead suspensionmixed with the preamplified amplicon library. The sample was thenpulse-spin and incubated at room temperature for 5 minutes. The tubecontaining the sample was placed on the magnetic rack for 2 minutes tocapture the beads. Once the solution cleared, the supernatant wascarefully discarded without disturbing the bead pellet. The desiredpreamplified amplicon library was now bound to the beads. Withoutremoving the tube from the magnetic rack, 200 microliters of freshlyprepared 70% ethanol was introduced into the sample. The sample wasincubated for 30 seconds while gently rotating the tube on the magneticrack. After the solution cleared, the supernatant was discarded withoutdisturbing the pellet. A second ethanol wash was performed and thesupernatant discarded. Any remaining ethanol was removed bypulse-spinning the tube and carefully removing residual ethanol whilenot disturbing the pellet. The pellet was air-dried for about 5 minutesat room temperature.

Once the tube was dry, the tube was removed from the magnetic rack and20 microliters of DNase/RNase Free Water was added (Life Technologies,CA, Part No. 600004). The tube was vortexed and pipetted to ensure thesample was mixed thoroughly. The sample was pulse-spin and placed on themagnetic rack for two minutes. After the solution cleared, thesupernatant containing the eluted DNA was transferred to a new tube.

Phosphorylate the Amplicons

To the eluted DNA (˜20 microliters), 3 microliters of DNA ligase buffer(Invitrogen, Catalog No. 15224041), 2 microliters dNTP mix, and 2microliters of FuP reagent were added. The reaction mixture was mixedthoroughly to ensure uniformity and incubated at 37° C. for 10 minutes.

Ligate Adapters to the Amplicons and Purify the Ligated Amplicons

After incubation, the reaction mixture proceeded directly to a ligationstep. Here, the reaction mixture now containing the phosphorylatedamplicon library was combined with 1 microliter of A/P1 Adaptersadapters(20 μm each) (sold as a component of the Ion Fragment Library Kit, LifeTechnologies, Part No. 4466464) and 1 microliter of DNA ligase (sold asa component of the Ion Fragment Library Kit, Life Technologies, Part No.4466464), and incubated at room temperature for 30 minutes.

After the incubation step, 52 microliters (1.8× sample volume) ofAgenCourt® AMPure® Reagent (Beckman Coulter, CA) was added to theligated DNA. The mixture was pipetted thoroughly to mix the beadsuspension with the ligated DNA. The mixture was pulse-spin andincubated at room temperature for 5 minutes. The samples underwentanother pulse-spin and were placed on a magnetic rack such as aDynaMag™-2 spin magnet (Life Technologies, CA, Part No. 123-21D) for twominutes. After the solution had cleared, the supernatant was discarded.Without removing the tube from the magnetic rack, 200 microliters offreshly prepared 70% ethanol was introduced into the sample. The samplewas incubated for 30 seconds while gently rotating the tube on themagnetic rack. After the solution cleared, the supernatant was discardedwithout disturbing the pellet. A second ethanol wash was performed andthe supernatant discarded. Any remaining ethanol was removed bypulse-spinning the tube and carefully removing residual ethanol whilenot disturbing the pellet. The pellet was air-dried for about 5 minutesat room temperature.

The pellet was resuspended in 20 microliters of DNase/RNase Free Water(Life Technologies, CA, Part No. 600004) and vortexed to ensure thesample was mixed thoroughly. The sample was pulse-spin and placed on themagnetic rack for two minutes. After the solution cleared, thesupernatant containing the ligated DNA was transferred to a new Lobindtube (Eppendorf, Part No. 022431021).

Nick Translate and Amplify the Amplicon Library and Purify the Library

The ligated DNA (˜20 microliters) was combined with 76 microliters ofPlatinum® PCR SuperMix High Fidelity (Life Technologies, CA, Part No.12532-016, sold as a component of the Ion Fragment Library Kit, LifeTechnologies, Part No. 4466464) and 4 microliters of LibraryAmplification Primer Mix (5 μM each) (Life Technologies, CA, Part No.602-1068-01, sold as a component of the Ion Fragment Library Kit, LifeTechnologies, Part No. 4466464), the mixture was pipetted thoroughly toensure a uniformed solution. The solution was applied to a single wellof a 96-well PCR plate and sealed. The plate was loaded into a thermalcycler (GeneAmp® PCR system 9700 Dual 96-well thermal cycler (LifeTechnologies, CA, Part No. N8050200 and 4314445)) and run on thefollowing temperate profile to generate the final amplicon library.

A nick-translation was performed at 72° C. for 1 minute, followed by anenzyme activation stage at 98° C. for 2 minutes, followed by 5-10 cyclesof denaturing at 98° C. for 15 seconds and an annealing and extendingstage at 60° C. for 1 minute. After cycling, the final amplicon librarywas held at 4° C. until proceeding to the final purification stepoutlined below.

In a 1.5 ml LoBind tube (Eppendorf, Part No. 022431021), the finalamplicon library (˜100 microliters) was combined with 180 microliters(1.8× sample volume) of Agencourt® AMPure® XP reagent (Beckman Coulter,CA). The bead suspension was pipetted up and down to thoroughly mix thebead suspension with the final amplicon library. The sample was thenpulse-spin and incubated for 5 minutes at room temperature.

The tube containing the final amplicon library was placed on a magneticrack such as a DynaMag™-2 spin magnet (Life Technologies, CA, Part No.123-21D) for 2 minutes to capture the beads. Once the solution cleared,the supernatant was carefully discarded without disturbing the beadpellet. Without removing the tube from the magnetic rack, 400microliters of freshly prepared 70% ethanol was introduced into thesample. The sample was incubated for 30 seconds while gently rotatingthe tube on the magnetic rack. After the solution cleared, thesupernatant was discarded without disturbing the pellet. A secondethanol wash was performed and the supernatant discarded. Any remainingethanol was removed by pulse-spinning the tube and carefully removingresidual ethanol while not disturbing the pellet. The pellet wasair-dried for about 5 minutes at room temperature.

Once the tube was dry, the tube was removed from the magnetic rack and20 microliters of Low TE was added (Life Technologies, CA, Part No.602-1066-01). The tube was pipetted and vortexed to ensure the samplewas mixed thoroughly. The sample was pulse-spin and placed on themagnetic rack for two minutes. After the solution cleared, thesupernatant containing the final amplicon library was transferred to anew Lobind tube (Eppendorf, Part No. 022431021).

Assess the Library Size Distribution and Determine the Template DilutionFactor

The final amplicon library was quantitated to determine the librarydilution (Template Dilution Factor) that results in a concentrationwithin the optimized target range for Template Preparation (e.g.,PCR-mediated addition of library molecules onto Ion Sphere™ Particles).The final amplicon library is typically quantitated for downstreamTemplate Preparation procedure using an Ion Library Quantitation Kit(qPCR) (Life Technologies, Part No. 4468802) and/or a Bioanalyzer™(Agilent Technologies, Agilent 2100 Bioanalyzer) to determine the molarconcentration of the amplicon library, from which the Template DilutionFactor is calculated. For example, instructions to determine theTemplate Dilution Factor by quantitative real-time PCR (qPCR) can befound in the Ion Library Quantitation Kit User Guide (Life Technologies,Part No. 4468986), hereby incorporated by reference in its entirety.

In this example, 1 microliter of the final amplicon library preparationwas analyzed on the 2100 Bioanalyzer™ with an Agilent High SensitivityDNA Kit (Agilent Technologies, Part No. 5067-4626) to generate peaks inthe 135-205 bp size range and at a concentration of about 5×10⁹ copiesper microliter.

Proceed to Template Preparation

An aliquot of the final library was used to prepare DNA templates thatwere clonally amplified on Ion Sphere™ Particles using emulsion PCR(emPCR). The preparation of template in the instant example was preparedaccording to the manufacturer's instructions using an Ion XpressTemplate Kit (Life Technologies, Part No. 4466457), hereby incorporatedby reference in its entirety. Once template-positive Ion SphereParticles were enriched, an aliquot of the Ion Spheres were loaded ontoan Ion 314™ Chip (Life Technologies, Part No. 4462923) as described inthe Ion Sequencing User Guide (Part No. 4467391), hereby incorporated inits entirety, and subjected to analysis and sequencing as described inthe Ion Torrent PGM Sequencer User Guide (Life Technologies, Part No.4462917), hereby incorporated in its entirety. The data obtained fromthis example is provided in FIG. 5.

Example 2 Optimizing Multiplex Reactions to Minimize GC Bias

This example demonstrates that optimizing multiplex PCR reactionsperformed under the guidance of the exemplary library amplificationprocess described in Example 1 help to minimize GC bias.

In this example, a low-plex sample containing 94 primer pairs oftarget-specific primers and a high-plex sample containing 380 primerpairs of target-specific primers were prepared from across the humangenome. Each low-plex and high plex sample was subjected to libraryamplification as described in Example 1. The melting temperature ofamplicon products was observed to correlate with GC content of theamplicon assay (FIG. 4A-4H). The high multiplex (384-plex) sample withhigh GC content (i.e., 80%) was observed to have a higher meltingtemperature than the corresponding 94-plex sample (FIG. 4A-4H).Additionally, high-plex samples with highest GC content (75% and 80%)were observed to possess a single predominant peak, the amplicon product(connected to X-axis via dotted line), in contrast to other non-specificamplification products (observed as additional peaks and noise acrossthe X-axis). The low-plex samples containing highest GC content (75% and80%) were found to form multiple peaks along the X-axis suggesting theformation of a substantial amount of non-specific amplification productsin addition to the desired amplicons. This example demonstrates thathigh-multiplex PCR reactions (394-plex) can be successfully optimized,for example through primer design, to increase the melting temperatureof the desired amplicon product, increase the amount of ampliconspecific product generated, and decrease the formation of amplificationartifacts.

Example 3 Optimizing Primer Design to Reduce Non-Specific Amplification

This example demonstrates that target-specific primers containingmodified nucleotides generated substantially fewer primer-dimers than acorresponding non-modified primer pool. In this example, a population ofnon-modified primer pairs were prepared using conventional methods andsubjected to the library amplification process as described inExample 1. A corresponding primer set containing modified nucleotideswere prepared and amplified as described in Example 1. The amount ofamplicon product and non-specific amplification product generated foreach primer pool was compared. The amount of primer-dimers observed inthe non-modified primer pool was found to greatly exceed the amount ofprimer-dimers in the corresponding modified primer pool (FIGS. 3A and3B). The production of amplification artifacts can cause significant, ifnot permanent, amplification issues in downstream amplification eventssuch as those commonly used in next-generating sequencing assays. Theuse of modified primers as disclosed herein can be used to alleviatethese issues and decrease the output of amplification artifacts permultiplex reaction.

Appendix A discloses the general state of the art for traditionalmultiplex PCR methods and highlights some of the disadvantages of thesemethods. Appendix A also discloses some of the high priority genesinitially evaluated as targets for the HSM (hotspot mutation panel).Appendix A provides a schematic outlining the workflow of the multiplexPCR methods disclosed herein. The Appendix also provides a graph showinga reduction in non-specific amplification products using the methods andtarget-specific primers according to the disclosure as compared tostandard multiplex primers (without a cleavable group). Appendix A alsodescribes an exemplary method for obtaining DNA from an FFPE sample, forexample using an Epicentre Quick Extract protocol. Results from studiesperformed using the exemplary methods and target-specific primersdisclosed herein using Ion Torrent 314, 316, and 318 chips wereperformed generating exemplary data regarding number of reads, coverageand total number of amplicons amenable to each chip type are provided inAppendix A.

Example 4 Generation of a 384-Plex (Multiplex) PCR Reaction

In this example, a multiplex polymerase chain reaction was performed toamplify 384 amplicons across genomic DNA. A primer pool containingmodified forward and reverse primer pairs was cycled with genomic DNAand subjected to library amplification as described in Example 1. Thedata obtained from 3 individual runs on an Ion Torrent PGM™ Sequencerwas averaged and is presented in FIG. 5. The data shows an average readrate of 1000 per amplicon.

Example 5 Effect of Forward and Reverse Primers on the Coverage of a384-Plex Multiplex PCR Reaction

In this example, a multiplex polymerase chain reaction was performed toamplify 411 amplicons across genomic DNA. The primer pool for theamplification reaction contained modified forward and reverse primersand was subjected to library amplification as described in Example 1.The data obtained from a single run on an Ion Torrent PGM™ Sequencershows the average read rate of about 400 for both the reverse andforward modified primers (FIGS. 6A and 6B).

Example 6 Reproducibility of 384-Multiplex Reactions

In this example, a multiplex polymerase chain reaction was performed toamplify 384 amplicons across genomic DNA. The primer pool containedmodified forward and reverse primer pairs and was subjected to libraryamplification as described in Example 1. The data obtained from 7individual runs on the Ion Torrent PGM™ Sequencer was averaged and ispresented in FIG. 7. The data shows an average read rate of about 400per amplicon.

Example 7 Reproducibility of 411-Multiplex Reactions

In this example, a multiplex polymerase chain reaction was performed toamplify 411 amplicons across genomic DNA. The primer pool containedmodified forward and reverse primer pairs and was subjected to libraryamplification as described in Example 1. The data obtained from 7individual runs on the Ion Torrent PGM™ Sequencer was averaged and ispresented in FIG. 8. The data shows an average read rate of about 400per amplicon.

Example 8 FFPE Samples as Amendable Substrates for Low Multiplex PCR

In this example, a multiplex polymerase chain reaction was performed toamplify 96 amplicons from a Fresh-Frozen Paraffin-Embedded (FFPE)sample. Fresh-Frozen and FFPE samples are often problematic foramplification processes due to the small amount of DNA that can beextracted from such samples. Additionally, because of the harsh chemicaltreatment required to preserve these samples, the quality of DNAextracted from such a sample is generally very poor. DNA was extractedfrom a FFPE sample and loaded onto an agarose gel for visualization(lanes 1 and 3) or subjected to multiplex PCR and library amplificationas described in Example 1 and then loaded onto the agarose gel forvisualization (lanes 2 and 4). A 96-plex PCR reaction and libraryamplification was performed on 10 ng of FFPE DNA as described inExample 1. FIG. 9 shows a photograph of the agarose electrophoresis gelincluding FFPE DNA prior to and after library amplification as describedin Example 1.

Example 9 FFPE Samples as Amenable Substrates for High Multiplex PCR

In this example, a multiplex polymerase chain reaction was performed toamplify 384 amplicons from a Fresh-Frozen Paraffin-Embedded (FFPE)sample. As discussed in Example 8, Fresh-Frozen and FFPE samples areoften problematic for amplification due to low extracted DNA quantityand quality. In this example, 10 ng of DNA was extracted from a FFPEsample and subjected to a 384-plex PCR reaction and libraryamplification as described by Example 1. The data obtained from the IonTorrent PGM™ Sequencer was averaged and is presented in FIG. 10. Thedata shows an average read rate of about 400 per amplicon. Furtheranalysis of the read rate for some of the modified forward and reverseprimer pairs used in the 384-plex reaction is presented in FIGS. 11A and11B.

Example 10 Detection of Variants in Control Mix of KRAS Codon 12 andCodon 13 Mutants

In this example, a sample of DNA containing spiked amounts of mutationsof the KRAS gene at codon 12 and codon 13 were provided as a blind testsample. The sample was amplified using target-specific modified primerpairs developed for the KRAS gene. The primer pool and samples wereamplified as described in Example 1. The amplified library was preparedas a template and enriched. The enriched template was applied to an Ion314™ Chip and analyzed using an Ion Torrent PGM™ Sequencer as describedin Example 1. The data from 3 individual runs was averaged and isprovided as FIG. 12. The amplicon products generated during the libraryamplification process correspond to the expected level of mutationspresent in the control DNA sample for the KRAS gene at codon 12 andcodon 13.

Example 11 Resequencing the Cystic Fibrosis Transmembrane ConductanceRegulator (CFTR) Gene Coding Region

The CFTR protein is encoded by the CFTR gene which is approximately 200kbp, spanning 27 exons. CFTR is an ABC transporter-class ion channelthat transports chloride and thiocyanate ions across epithelial cellmembranes. Mutations of the CFTR gene affect functioning of the chlorideion channels in these cell membranes, leading to cystic fibrosis andcongenital absence of the vas deferens. As a consequence, a male carrierof the mutation in the CFTR gene can be infertile so the detection ofthe defect is key in IVD considerations.

In this example, genomic DNA (gDNA) was obtained containing mutations ofthe CFTR gene. The gDNA was prepared for library amplification, templatepreparation and applied to Ion Torrent 314™ Chip as described inExample 1. Over 8947 bases were amplified during the multiplex PCRreaction. The resulting amplicon library contained 34 amplicons with anaverage amplicon length of 104 bp. Sequence analysis of the ampliconlibrary was conducted against the CFTR gene sequence reference: NCBIgi|287325315|ref|NG_(—)016465.1|, 195703 bp

The generated amplicon library was used to evaluate detection of theCFTR gene mutations in the sample. It was found that the libraryamplification process detected and identified 5 point mutations and 1insertion/deletion at low coverage (4,001 reads) (FIG. 13 (Part I-PartIII). The naming system for the 6 mutations is as follows:

Naming system: CFMD (UMD)

c.869+11C>T (1001+11C/T)

c.1408A>G (M470V)

c.2562T>G (2694T/G)

c.4389G>A (4521G/A)

c.1-8G>C (125G/C)

c.1521_(—)1523deICTT (ΔF508)

Most resequencing assays are based on amplicons that are greater than100 bp. This example demonstrates the applicability and amenability ofthe library amplification process exemplified in Example 1, to generateamplicons of a length necessary for resequencing assays. The examplealso shows the accuracy of the Ion Torrent PGM™ Sequencer to correctlyidentify point mutations and insertions/deletion mutations in a givensample.

Example 12 Generation of Target-Specific Primers

Table 2 provides a list of target-specific forward and reverse uracilcontaining primers that were used in the above examples to amplifytarget-specific regions of gDNA or DNA extracted from samples, such asFFPE samples. Table 2 provides the chromosome location of each primerpair, the nucleotide sequence of each forward and reverse primer in eachprimer pair. Table 2 also provides the coordinates of the 5′ end of theupstream/forward primer, the length of each amplicon, the correspondingamplicon nucleotide sequence, the length of each forward and reverseprimer, and the T_(m) for each forward or reverse primer. Tables 3a-3dalso provide a list of distinct target-specific forward and reverseprimer pairs that were used in example 13 to amplify target-specificregions of gDNA or DNA extracted from FFPE samples from the genes ofTable 1 that are associated with cancer. The following primer pair wasalso included for coverage of a mutation in the AKT1 gene (c.49G>A).Forward primer: GCCGCCAGGUCTTGATGUA and reverse primer:GCACAUCTGTCCUGGCACA.

Example 13 Alternate Library Protocol

PCR Amplify Genomic DNA Targets

In this example, a multiplex polymerase chain reaction was performed toamplify multiple individual amplicons across a genomic DNA sample orFFPE sample. A representative list of genes associated with cancers thatwere incorporated for investigation while synthesizing the primer poolis provided in Table 1. Each primer pair in the primer pool was designedto contain at least one uracil nucleotide in each of the forward andreverse primer (Tables 3a-3d). Each primer pair was also designed toselectively hybridize to, and promote amplification of a specific geneor gene fragment of the genomic DNA sample to reduce formation ofnon-specific amplification products.

To a single well of a 96-well PCR plate was added 4 microliters of 5×Primer Pool (containing the primer pairs) at a concentration of 250 nmeach in TE, 10 ng of genomic DNA and 10 microliters of an amplificationreaction mixture (2× Stoffel HiFi Master Mix) that can include glycerol,dNTPs and Stoffel fragment of Amplitaq® DNA Polymerase (Invitrogen,Catalog No. N8080038) to a final volume of 20 microliters with nucleasefree water (Life Technologies, CA, Part No. 600004).

The PCR plate was sealed and loaded into a thermal cycler (GeneAmp® PCRsystem 9700 Dual 96-well thermal cycler (Life Technologies, CA, Part No.N8050200 and 4314445)) and run on the following temperature profile togenerate the preamplified amplicon library. Variation to the number ofcycles was performed based on the total plexy of the reaction mixtureunder investigation. For example, a plexy of 48-96 was run for 17cycles; a plexy of 97-192 was run for 16 cycles; a plexy of 193-384 wasrun for 15 cycles; a plexy of 385-768 was run for 14 cycles; a plexy of769-1536 was run for 13 cycles. Additionally, for reaction mixturescontaining barcodes or pooled reaction mixtures the number of cycles waslowered by one or more additional cycles. For example, 2-3 barcodes persample were subtracted by one cycle; 4-8 barcodes per sample weresubtracted by 2 cycles, and 9-16 barcodes were subtracted by 3 cycles.An initial holding stage was performed at 99° C. for 2 minutes, followedby X cycles (as determined above) of denaturing at 99° C. for 15 secondsand an annealing and extending stage at 60° C. for 4 minutes. Aftercycling, the preamplified amplicon library was held at 4° C. untilproceeding to the digestion and phosphorylation step outlined below.

Digest/Phosphorylate/Heat Kill the Amplicons

To the preamplified library (˜20 microliters), 2 microliters of FuPareagent was added. Typically, the FuPa reagent comprises one or moreuracil degradable enzymes such as UDG, FPG and the like, a DNApolymerase such as Pol I, T4PNK, Klenow and the like, and an antibodysuch as an anti-Taq antibody. In this example, the relative amount ofeach enzymatic component in the FuPa reagent was 1:1:1:1 but can bevaried according to the number of required cycles, variations to thetemperature profile, etc., as can be determined by one of ordinary skillin the art. The PCR place was sealed and loaded into a thermal cycler(GeneAmp® PCR system 9700 Dual 96-well thermal cycler (LifeTechnologies, CA, Part No. N8050200 and 4314445)) and run on thefollowing temperature profile. An initial holding stage was performed at37° C. for 5 minutes, followed by 55° C. for 10 minutes and then 60° C.for 20 minutes. After cycling, the preamplified amplicon library washeld at 4° C. until proceeding to the ligation/nick translation stepoutlined below.

Ligate Adapters to the Amplicons and Nick Translate

After phosphorylation, the amplicon preamplification library (˜22 μl)proceeded directly to a ligation step. In this example, thepreamplification library now containing the phosphorylated ampliconlibrary was combined with 1 microliter of A/P1 Adaptersadapters (20 μmeach) (sold as a component of the Ion Fragment Library Kit, LifeTechnologies, Part No. 4466464), 2 microliters of 10× ligation bufferand 1 microliter of DNA ligase (sold as components of the Ion FragmentLibrary Kit, Life Technologies, Part No. 4466464). The PCR plate wassealed and loaded into a thermal cycler (GeneAmp® PCR system 9700 Dual96-well thermal cycler (Life Technologies, CA, Part No. N8050200 and4314445)) and run on the following temperature profile. An initialholding stage was performed at 22° C. for 30 minutes, followed by 65° C.for 10 minutes and then held at 4° C. until proceeding to the next step.

If the amplicon library is to contain barcodes (for example Ion DNABarcoding 1-16 kit, Life Technologies, Part No. 4468654, incorporatedherein in its entirety), the barcodes can be added at this step to thePCR plate essentially according to the manufacturer's instructions priorto proceeding to the next step

1.8× AMPure XP Purification

1.8× sample volume of AgenCourt® AMPure® Reagent (Beckman Coulter, CA)was added to the ligated DNA. The mixture was mixed and incubated atroom temperature for 5 minutes. After the solution had cleared, thesupernatant was discarded. An ethanol wash was performed and thesupernatant discarded. Any remaining ethanol was removed and the pelletwas air-dried for about 5 minutes at room temperature. The pellet wasresuspended in 20 microliters of DNase/RNase Free Water (LifeTechnologies, CA, Part No. 600004). In some instances, an optionallibrary amplification step can be performed on the amplicon library, asoutlined below.

Nick Translate and Amplify the Amplicon Library and Purify the Library

The ligated DNA (˜20 microliters) was combined with 78 microliters ofPlatinum® PCR SuperMix High Fidelity (Life Technologies, CA, Part No.12532-016, sold as a component of the Ion Fragment Library Kit, LifeTechnologies, Part No. 4466464) and 4 microliters of LibraryAmplification Primer Mix (5 μM each) (Life Technologies, CA, Part No.602-1068-01, sold as a component of the Ion Fragment Library Kit, LifeTechnologies, Part No. 4466464). The solution was applied to a singlewell of a 96-well PCR plate and sealed. The plate was loaded into athermal cycler (GeneAmp® PCR system 9700 Dual 96-well thermal cycler(Life Technologies, CA, Part No. N8050200 and 4314445)) and run on thefollowing temperate profile to generate the final amplicon library.

A nick-translation was performed at 72° C. for 1 minute, followed by anenzyme activation stage at 98° C. for 2 minutes, followed by 7 cycles ofdenaturing at 98° C. for 15 seconds and an annealing and extending stageat 60° C. for 1 minute. After cycling, the final amplicon library washeld at 4° C. until proceeding to the final purification step outlinedbelow.

The final amplicon library (˜100 microliters) was combined with 1.8×sample volume of Agencourt® AMPure® XP reagent (Beckman Coulter, CA).The mixture was then incubated for 5 minutes at room temperature. ThePCR plate containing the final amplicon library was washed with 70%ethanol and the supernatant discarded. Any remaining ethanol was removedand air-dried for about 5 minutes at room temperature. Once dry, thelibrary was resuspended in 20 microliters of Low TE (Life Technologies,CA, Part No. 602-1066-01).

Assess the Library Size Distribution and Determine the Template DilutionFactor

The final amplicon library was quantitated to determine the librarydilution (Template Dilution Factor) that results in a concentrationwithin the optimized target range for Template Preparation (e.g.,PCR-mediated addition of library molecules onto Ion Sphere™ Particles).The final amplicon library is typically quantitated for downstreamTemplate Preparation procedure using an Ion Library Quantitation Kit(qPCR) (Life Technologies, Part No. 4468802) and/or a Bioanalyzer™(Agilent Technologies, Agilent 2100 Bioanalyzer) to determine the molarconcentration of the amplicon library, from which the Template DilutionFactor is calculated. For example, instructions to determine theTemplate Dilution Factor by quantitative real-time PCR (qPCR) can befound in the Ion Library Quantitation Kit User Guide (Life Technologies,Part No. 4468986), hereby incorporated by reference in its entirety.

In this example, 1 microliter of the final amplicon library preparationwas analyzed on the 2100 Bioanalyzer™ with an Agilent High SensitivityDNA Kit (Agilent Technologies, Part No. 5067-4626) to generate peaks inthe 135-205 bp size range and at a concentration of about 5×10⁹ copiesper microliter.

Proceed to Template Preparation

An aliquot of the final library was used to prepare DNA templates thatwere clonally amplified on Ion Sphere™ Particles using emulsion PCR(emPCR). The preparation of template in the instant example was preparedaccording to the manufacturer's instructions using an Ion XpressTemplate Kit (Life Technologies, Part No. 4466457), hereby incorporatedby reference in its entirety. Once template-positive Ion SphereParticles were enriched, an aliquot of the Ion Spheres were loaded ontoan Ion 314™ Chip (Life Technologies, Part No. 4462923) as described inthe Ion Sequencing User Guide (Part No. 4467391), hereby incorporated inits entirety, and subjected to analysis and sequencing as described inthe Ion Torrent PGM Sequencer User Guide (Life Technologies, Part No.4462917), hereby incorporated in its entirety.

Example 14 Alternate Library Protocol

PCR Amplify Genomic DNA Targets

In this example, a multiplex polymerase chain reaction was performed toamplify multiple individual amplicons across a genomic DNA sample. Arepresentative list of genes associated with cancers that wereincorporated for investigation while synthesizing the primer pool isprovided in Table 1. Each primer pair in the primer pool was designed tocontain at least one uracil nucleotide in each of the forward andreverse primer (Table 2). Each primer pair was also designed toselectively hybridize to, and promote amplification of a specific geneor gene fragment of the genomic DNA sample to reduce formation ofnon-specific amplification products.

To a single well of a 96-well PCR plate was added 5 microliters of 4×HSMPrimer Pool (containing the primer pairs of Table 2) at a concentrationof 250 nm each in TE, 50 ng of genomic DNA and 10 microliters of anamplification reaction mixture (2× PreAmp HiFi Master Mix) that caninclude glycerol, dNTPs and a DNA Polymerase (such as Taq DNAPolymerase, Invitrogen, Catalog No. N8080038) to a final volume of 20microliters with nuclease free water (Life Technologies, CA, Part No.600004).

The PCR plate was sealed and loaded into a thermal cycler (GeneAmp® PCRsystem 9700 Dual 96-well thermal cycler (Life Technologies, CA, Part No.N8050200 and 4314445)) and run on the following temperature profile togenerate the preamplified amplicon library. An initial holding stage wasperformed at 98° C. for 2 minutes, followed by 16 cycles of denaturingat 98° C. for 15 seconds and an annealing and extending stage at 60° C.for 4 minutes. After cycling, the preamplified amplicon library was heldat 4° C. until proceeding to the purification step outlined below.

Purify the Amplicons from Input DNA and Primers

Two rounds of Agencourt® AMPure® XP Reagent (Beckman Coulter, CA)binding, wash, and elution at 0.6× and 1.2× volume ratios were found toremove genomic DNA and unbound or excess primers. In a 1.5 ml LoBindtube (Eppendorf, Part No. 022431021), the preamplified amplicon library(20 microliters) was combined with 12 microliters (0.6× volumes) ofAgencourt® AMPure® XP reagent (Beckman Coulter, CA). The bead suspensionwas pipetted up and down to thoroughly mix the bead suspension with thepreamplified amplicon library. The sample was then pulse-spin andincubated for 5 minutes at room temperature.

The tube containing the sample was placed on a magnetic rack such as aDynaMag™-2 spin magnet (Life Technologies, CA, Part No. 123-21D) for 2minutes to capture the beads. Once the solution cleared, the supernatantwas transferred to a new tube, where 24 microliters (1.2× volume) ofAgenCourt® AMPure® XP beads (Beckman Coulter, CA) were added to thesupernatant. The mixture was pipetted to ensure the bead suspensionmixed with the preamplified amplicon library. The sample was thenpulse-spin and incubated at room temperature for 5 minutes. The tubecontaining the sample was placed on the magnetic rack for 2 minutes tocapture the beads. Once the solution cleared, the supernatant wascarefully discarded without disturbing the bead pellet. The desiredpreamplified amplicon library was now bound to the beads. Withoutremoving the tube from the magnetic rack, 200 microliters of freshlyprepared 70% ethanol was introduced into the sample. The sample wasincubated for 30 seconds while gently rotating the tube on the magneticrack. After the solution cleared, the supernatant was discarded withoutdisturbing the pellet. A second ethanol wash was performed and thesupernatant discarded. Any remaining ethanol was removed bypulse-spinning the tube and carefully removing residual ethanol whilenot disturbing the pellet. The pellet was air-dried for about 5 minutesat room temperature.

Once the tube was dry, the tube was removed from the magnetic rack and20 microliters of DNase/RNase Free Water was added (Life Technologies,CA, Part No. 600004). The tube was vortexed and pipetted to ensure thesample was mixed thoroughly. The sample was pulse-spin and placed on themagnetic rack for two minutes. After the solution cleared, thesupernatant containing the eluted DNA was transferred to a new tube.

Phosphorylate the Amplicons

To the eluted DNA (˜20 microliters), 3 microliters of DNA ligase buffer(Invitrogen, Catalog No. 15224041), 2 microliters dNTP mix (10 mm), and2 microliters of FuP reagent were added. The reaction mixture was mixedthoroughly to ensure uniformity and incubated at 37° C. for 10 minutes.

Ligate Adapters to the Amplicons and Purify the Ligated Amplicons

After incubation, the reaction mixture proceeded directly to a ligationstep. Here, the reaction mixture now containing the phosphorylatedamplicon library was combined with 1 microliter of A/P1 adapters (20 μmeach) (sold as a component of the Ion Fragment Library Kit, LifeTechnologies, Part No. 4466464) and 1 microliter of DNA ligase (sold asa component of the Ion Fragment Library Kit, Life Technologies, Part No.4466464), and incubated at room temperature for 30 minutes.

After the incubation step, 52 microliters (1.8× sample volume) ofAgenCourt® AMPure® Reagent (Beckman Coulter, CA) was added to theligated DNA. The mixture was pipetted thoroughly to mix the beadsuspension with the ligated DNA. The mixture was pulse-spin andincubated at room temperature for 5 minutes. The samples underwentanother pulse-spin and were placed on a magnetic rack such as aDynaMag™-2 spin magnet (Life Technologies, CA, Part No. 123-21D) for twominutes. After the solution had cleared, the supernatant was discarded.Without removing the tube from the magnetic rack, 200 microliters offreshly prepared 70% ethanol was introduced into the sample. The samplewas incubated for 30 seconds while gently rotating the tube on themagnetic rack. After the solution cleared, the supernatant was discardedwithout disturbing the pellet. A second ethanol wash was performed andthe supernatant discarded. Any remaining ethanol was removed bypulse-spinning the tube and carefully removing residual ethanol whilenot disturbing the pellet. The pellet was air-dried for about 5 minutesat room temperature.

The pellet was resuspended in 20 microliters of DNase/RNase Free Water(Life Technologies, CA, Part No. 600004) and vortexed to ensure thesample was mixed thoroughly. The sample was pulse-spin and placed on themagnetic rack for two minutes. After the solution cleared, thesupernatant containing the ligated DNA was transferred to a new Lobindtube (Eppendorf, Part No. 022431021).

Nick Translate and Amplify the Amplicon Library and Purify the Library

The ligated DNA (˜20 microliters) was combined with 76 microliters ofPlatinum® PCR SuperMix High Fidelity (Life Technologies, CA, Part No.12532-016, sold as a component of the Ion Fragment Library Kit, LifeTechnologies, Part No. 4466464) and 4 microliters of LibraryAmplification Primer Mix (5 μM each) (Life Technologies, CA, Part No.602-1068-01, sold as a component of the Ion Fragment Library Kit, LifeTechnologies, Part No. 4466464), the mixture was pipetted thoroughly toensure a uniformed solution. The solution was applied to a single wellof a 96-well PCR plate and sealed. The plate was loaded into a thermalcycler (GeneAmp® PCR system 9700 Dual 96-well thermal cycler (LifeTechnologies, CA, Part No. N8050200 and 4314445)) and run on thefollowing temperate profile to generate the final amplicon library.

A nick-translation was performed at 72° C. for 1 minute, followed by anenzyme activation stage at 98° C. for 2 minutes, followed by 6 cycles ofdenaturing at 98° C. for 15 seconds and an annealing and extending stageat 60° C. for 1 minute. After cycling, the final amplicon library washeld at 4° C. until proceeding to the final purification step outlinedbelow.

In a 1.5 ml LoBind tube (Eppendorf, Part No. 022431021), the finalamplicon library (˜100 microliters) was combined with 180 microliters(1.8× sample volume) of Agencourt® AMPure® XP reagent (Beckman Coulter,CA). The bead suspension was pipetted up and down to thoroughly mix thebead suspension with the final amplicon library. The sample was thenpulse-spin and incubated for 5 minutes at room temperature.

The tube containing the final amplicon library was placed on a magneticrack such as a DynaMag™-2 spin magnet (Life Technologies, CA, Part No.123-21D) for 2 minutes to capture the beads. Once the solution cleared,the supernatant was carefully discarded without disturbing the beadpellet. Without removing the tube from the magnetic rack, 400microliters of freshly prepared 70% ethanol was introduced into thesample. The sample was incubated for 30 seconds while gently rotatingthe tube on the magnetic rack. After the solution cleared, thesupernatant was discarded without disturbing the pellet. A secondethanol wash was performed and the supernatant discarded. Any remainingethanol was removed by pulse-spinning the tube and carefully removingresidual ethanol while not disturbing the pellet. The pellet wasair-dried for about 5 minutes at room temperature.

Once the tube was dry, the tube was removed from the magnetic rack and20 microliters of Low TE was added (Life Technologies, CA, Part No.602-1066-01). The tube was pipetted and vortexed to ensure the samplewas mixed thoroughly. The sample was pulse-spin and placed on themagnetic rack for two minutes. After the solution cleared, thesupernatant containing the final amplicon library was transferred to anew Lobind tube (Eppendorf, Part No. 022431021).

Assess the Library Size Distribution and Determine the Template DilutionFactor

The final amplicon library was quantitated to determine the librarydilution (Template Dilution Factor) that results in a concentrationwithin the optimized target range for Template Preparation (e.g.,PCR-mediated addition of library molecules onto Ion Sphere™ Particles).The final amplicon library is typically quantitated for downstreamTemplate Preparation procedure using an Ion Library Quantitation Kit(qPCR) (Life Technologies, Part No. 4468802) and/or a Bioanalyzer™(Agilent Technologies, Agilent 2100 Bioanalyzer) to determine the molarconcentration of the amplicon library, from which the Template DilutionFactor is calculated. For example, instructions to determine theTemplate Dilution Factor by quantitative real-time PCR (qPCR) can befound in the Ion Library Quantitation Kit User Guide (Life Technologies,Part No. 4468986), hereby incorporated by reference in its entirety.

In this example, 1 microliter of the final amplicon library preparationwas analyzed on the 2100 Bioanalyzer™ with an Agilent High SensitivityDNA Kit (Agilent Technologies, Part No. 5067-4626).

Proceed to Template Preparation

An aliquot of the final library was used to prepare DNA templates thatwere clonally amplified on Ion Sphere™ Particles using emulsion PCR(emPCR). The preparation of template in the instant example was preparedaccording to the manufacturer's instructions using an Ion XpressTemplate Kit (Life Technologies, Part No. 4466457), hereby incorporatedby reference in its entirety. Once template-positive Ion SphereParticles were enriched, an aliquot of the Ion Spheres were loaded ontoan Ion 314™ Chip (Life Technologies, Part No. 4462923) as described inthe Ion Sequencing User Guide (Part No. 4467391), hereby incorporated inits entirety, and subjected to analysis and sequencing as described inthe Ion Torrent PGM Sequencer User Guide (Life Technologies, Part No.4462917), hereby incorporated in its entirety.

Example 15 Alternate Library Protocol

PCR Amplify Genomic DNA Targets

In this example, a multiplex polymerase chain reaction was performed toamplify multiple individual amplicons across a genomic DNA sample. Arepresentative list of genes associated with cancers that wereincorporated for investigation while synthesizing the primer pool isprovided in Table 1. Each primer pair in the primer pool was designed tocontain at least one uracil nucleotide in each of the forward andreverse primer (Table 2). Each primer pair was also designed toselectively hybridize to, and promote amplification of a specific geneor gene fragment of the genomic DNA sample to reduce formation ofnon-specific amplification products.

To a single well of a 96-well PCR plate was added 10 microliters of 2×Primer Pool (containing the primer pairs of Table 2) at a concentrationof 250 nm each, 10 ng of genomic DNA and 4 microliters of anamplification reaction mixture (5× Ion Ampliseq Master Mix) that caninclude glycerol, dNTPs and a DNA polymerase (for example, Stoffelfragment of Amplitaq® DNA Polymerase (Invitrogen Catalog No. N8080038))to a final volume of 20 microliters with nuclease free water (LifeTechnologies, CA, Part No. 600004).

The PCR plate was sealed and loaded into a thermal cycler (GeneAmp® PCRsystem 9700 Dual 96-well thermal cycler (Life Technologies, CA, Part No.N8050200 and 4314445)) and run on the following temperature profile togenerate the preamplified amplicon library. Variation to the number ofcycles was performed based on the total plexy of the reaction mixtureunder investigation. For example, a plexy of 48-96 was run for 17cycles; a plexy of 97-192 was run for 16 cycles; a plexy of 193-384 wasrun for 15 cycles; a plexy of 385-768 was run for 14 cycles; a plexy of769-1536 was run for 13 cycles. Additionally, for reaction mixturescontaining barcodes or pooled reaction mixtures the number of cycles waslowered by one or more additional cycles. For example, 2-3 barcodes persample were subtracted by one cycle; 4-8 barcodes per sample weresubtracted by 2 cycles, and 9-16 barcodes were subtracted by 3 cycles.For samples that contain fragmented DNA, e.g., enzymatically digestedDNA, the number of cycles can be increased for up to 3 cycles. Forselective amplification of one or more DNA samples using a startinginput of 1 ng or less DNA, the number of cycles was increased by anadditional 3 cycles. An initial holding stage was performed at 99° C.for 2 minutes, followed by X cycles (as determined above) of denaturingat 99° C. for 15 seconds and an annealing and extending stage at 60° C.for 4 minutes. After cycling, the preamplified amplicon library was heldat 4° C. until proceeding to the purification step outlined below.

Digest/Phosphorylate/Heat Kill the Amplicons

To the preamplified library (˜20 microliters), 2 microliters of FuPareagent was added. Typically, the FuPa reagent comprises one or moreuracil degradable enzymes such as UDG, FPG and the like, a DNApolymerase such as Klenow fragment, and an antibody such as an anti-Taqantibody. In this example, the relative amount of each enzymaticcomponent in the FuPa reagent was 1:1:1:2 but can be varied according tothe number of required cycles, variations to the temperature profile,etc., as can be determined by one of ordinary skill in the art. The PCRplate was sealed and loaded into a thermal cycler (GeneAmp® PCR system9700 Dual 96-well thermal cycler (Life Technologies, CA, Part No.N8050200 and 4314445)) and run on the following temperature profile. Aninitial holding stage was performed at 37° C. for 10 minutes, followedby 55° C. for 10 minutes and then 60° C. for 20 minutes. After cycling,the preamplified amplicon library was held at 4° C. until proceeding tothe ligation/nick translation step outlined below.

Ligate Adapters to the Amplicons and Nick Translate

After phosphorylation, the amplicon preamplification library (˜22 μl)proceeded directly to a ligation step. In this example, thepreamplification library now containing the phosphorylated ampliconlibrary was combined with 2 microliters of A/P1 Adaptersadapters (5each) (sold as a component of the Ion Fragment Library Kit, LifeTechnologies, Part No. 4466464), 4 microliters of 7.5× ligation bufferand 2 microliters of T4 DNA ligase (5 u/μl). The PCR plate was sealedand loaded into a thermal cycler (GeneAmp® PCR system 9700 Dual 96-wellthermal cycler (Life Technologies, CA, Part No. N8050200 and 4314445))and run on the following temperature profile. An initial holding stagewas performed at 22° C. for 30 minutes, followed by 60° C. for 5 minutesand then held at 4° C. until proceeding to the next step.

If the amplicon library is to contain barcodes (for example Ion DNABarcoding 1-16 kit, Life Technologies, Part No. 4468654, incorporatedherein in its entirety), the barcodes are added at this step to the PCRplate essentially according to the manufacturer's instructions prior toproceeding to the next step. Optionally, all the samples or barcodes canbe pooled into a single tube at this step.

1.6× AMPure XP Purification

1.6× sample volume of AgenCourt® AMPure® Reagent (Beckman Coulter, CA)was added to the ligated DNA. The mixture was mixed and incubated atroom temperature for 5 minutes. An ethanol wash was performed and thesupernatant discarded. Any remaining ethanol was removed and air-driedfor about 5 minutes at room temperature. The dry tube containing thelibrary was resuspended in 20 microliters of Nuclease Free Water (LifeTechnologies, CA, Part No. 600004). In some instances, an optional nicktranslation/library amplification step can be performed on the ampliconlibrary, as outlined below.

Nick Translate and Amplify the Amplicon Library and Purify the Library

The ligated DNA (˜20 microliters) was combined with 78 microliters ofPlatinum® PCR SuperMix High Fidelity (Life Technologies, CA, Part No.12532-016, sold as a component of the Ion Fragment Library Kit, LifeTechnologies, Part No. 4466464) and 4 microliters of LibraryAmplification Primer Mix (5 μM each) (Life Technologies, CA, Part No.602-1068-01, sold as a component of the Ion Fragment Library Kit, LifeTechnologies, Part No. 4466464). The solution was applied to a singlewell of a 96-well PCR plate and sealed. The plate was loaded into athermal cycler (GeneAmp® PCR system 9700 Dual 96-well thermal cycler(Life Technologies, CA, Part No. N8050200 and 4314445)) and run on thefollowing temperate profile to generate the final amplicon library.

A nick-translation was performed at 72° C. for 1 minute, followed by anenzyme activation stage at 98° C. for 2 minutes, followed by 7 cycles ofdenaturing at 98° C. for 15 seconds and an annealing and extending stageat 60° C. for 1 minute. After cycling, the final amplicon library washeld at 4° C. until proceeding to the final purification step outlinedbelow.

The final amplicon library (˜100 microliters) was combined with 1.8×sample volume of Agencourt® AMPure® XP reagent (Beckman Coulter, CA).The mixture was then incubated for 5 minutes at room temperature. Thefinal amplicon library was washed with 70% ethanol and the supernatantdiscarded. Any remaining ethanol was removed and air-dried for about 5minutes at room temperature. Once dry, the library was resuspended in 20microliters of Low TE (Life Technologies, CA, Part No. 602-1066-01). Inthis example, 1 microliter of the final amplicon library preparation wasanalyzed on the 2100 Bioanalyzer™ with an Agilent High Sensitivity DNAKit (Agilent Technologies, Part No. 5067-4626).

Example 16

In this example, an amplicon library was prepared using 2946target-specific primer pairs. The primer pairs were prepared from thelist of genes in Tables 1 and 18. Each primer pair was designed toselectively hybridize and promote amplification of the selected targetedregion. The libraries were prepared according to Example 14 except thatthe primer pool was pooled into a single PCR tube, evaporated andresuspended in low TE and supplemented with 0.25 mM magnesium chlorideprior to incubating with DNA and the amplification reaction mixture. Inthis example, both high molecular weight DNA or mechanically sheared DNAwere applied as input DNA (10 ng input). The results from thissequencing experiment are provided in the Table below and demonstratedapproximately 91-98% of all reads were on target, greater than 99%target coverage at 1× when normalized to 100×, and greater than 98% baseaccuracy.

Plexity of PreAmp 2946 2946 Target DNA Sheared HMW Percent greater than0.01 mean 99.40% 99.29% reads per base Percent greater than 0.1 mean96.33% 95.16% reads per base Percent greater than 0.2 mean 92.26% 89.40%reads per base Percent no strand bias of all Amps 71.15% 72.98% Percentno strand bias of all Amps 73.31% 77.12% >100 reads Percent end to endread of on −1.00% −1.00% target reads Per base accuracy 98.34% 98.59%Percent of total reads mapped 99.10% 99.14% on target Percent wells withread 35.94% 31.23% Number of total reads 2277968 1979529 Number ofmapped reads 2257427 1962501 Number of reads on target 2094328 1941916Percent mapped reads on target 92.78% 98.95% Percent reads off target7.16% 1.04% Percent reads unmapped 0.90% 0.86% Bases in targetedreference 327815 327815 Bases covered (at least 1x) 326642 326656 Totalbase reads on target 220235195 206585756 Average base coverage depth671.83 630.19 Maximum base read depth 3070 2520 Average base read depth674.23 632.43 Std. Dev base read depth 431.42 466.91 Target coverage at1x 99.64% 99.65% Target coverage at 10x 99.23% 99.12% Target coverage at20x 98.77% 98.42% Target coverage at 10x - norm 100 96.33% 95.16%Percent end to end read of on −1.00% −1.00% target reads Percent forwardend to end read −1.00% −1.00% of on target reads Percent reverse end toend read −1.00% −1.00% of on target reads Coverage needed for 99percentile 52.2 48.96 base with at least 1x coverage Coverage needed for98 percentile 207.65 247.24 base with at least 10x coverage Coverageneeded for 95 percentile 145.33 203.58 base with at least 20x coverage

Example 17

In this example, an amplicon library was prepared using 6110target-specific primer pairs. The library corresponds to approximately450 cancer genes. The primer pairs were prepared from the list of genesin Tables 1 and 18. Each primer pair was designed to selectivelyhybridize and promote amplification of the selected targeted regionusing the target-specific selection criteria outline herein. Thelibraries were prepared according to Example 14 except that the primerpools were prepared as two tubes (3188-plex and 2946-plex) andsupplemented with 0.5 mM magnesium chloride. The PCR preamplificationcycling steps were also modified to increase the denaturing temperatureto 99° C., included an extending step of 72° C. for 5 minutes, after the60° C. annealing step, which was also extended to 10 minutes. Aftercycling, each tube containing the amplicon library was combined into asingle emulsion for emulsion PCR enrichment. The amplicon library wasused to prepare DNA templates that were clonally amplified on IonSphere™ Particles using emulsion PCR (emPCR). The preparation oftemplate in the instant example was prepared according to themanufacturer's instructions using an Ion Xpress Template Kit (LifeTechnologies, Part No. 4466457), hereby incorporated by reference in itsentirety. Once template-positive Ion Sphere Particles were enriched, analiquot of the Ion Spheres were loaded onto an Ion 316™ Chip (LifeTechnologies, Part No. 4466616) as described in the Ion Sequencing UserGuide (Part No. 4467391), hereby incorporated in its entirety, andsubjected to analysis and sequencing as described in the Ion Torrent PGMSequencer User Guide (Life Technologies, Part No. 4462917), herebyincorporated in its entirety.

In this example, high molecular weight DNA was applied as the input DNA(10 ng). The results from this experiment are provided in the Tablebelow and demonstrated approximately 95% of all reads were on target,approximately 95% of all reads (greater than 100 reads) showed no strandbias, and greater than 97% base accuracy.

Percent greater than 0.01 mean reads per base 81.58% Percent greaterthan 0.1 mean reads per base 75.40% Percent greater than 0.2 mean readsper base 69.73% Percent no strand bias of all bases 92.16% Percent nostrand bias of all Amps 72.16% Percent no strand bias of all Amps >100reads 95.06% Percent end to end read of on target reads −1.00% Per baseaccuracy 97.78% Percent of total reads mapped to hg19 98.05% Percentwells with read 34.51% Number of total reads 2190638 Number of mappedreads 2147835 Number of targets 6110 Number of reads on target 2098176Percent all reads on target 95.78% Percent mapped reads on target 97.69%Percent reads off target 2.27% Percent reads unmapped 1.95% Bases intargeted reference 677270 Bases covered (at least 1x) 556880 Total basereads on target 243058082 Average base coverage depth 358.88 Maximumbase read depth 2402 Average base read depth 436.45 Std. Dev base readdepth 371.45 Target coverage at 1x 82.22% Target coverage at 10x 80.45%Target coverage at 20x 78.41% Target coverage at 100x 66.15% Targetcoverage at 500x 29.11% Target coverage at 1x - norm 100 81.58% Targetcoverage at 10x - norm 100 75.40% Target coverage at 20x - norm 10069.73% Target coverage at 100x - norm 100 40.40% Target coverage at500x - norm 100 0.34% Percent end to end read of on target reads −1.00%Percent forward end to end read of on target reads −1.00% Percentreverse end to end read of on target reads −1.00% Coverage needed for 99percentile base with at least 865.36 1x coverage Coverage needed for 98percentile base with at least 8653.57 20x coverage Coverage needed for95 percentile base with at least 60574.98 350x coverage Percent priority1 design covered 0 Percent priority 9 design covered 0

Example 18

In this example, an amplicon library was prepared using approximately1500 target-specific primer pairs. The primer pairs were prepared fromgenes in Table 1. Each primer pair was designed to selectively hybridizeand promote amplification of the selected targeted region using thetarget-specific primer selection criteria outlined herein. The librarieswere prepared according to Example 15 except that the number ofpreamplification PCR cycles was amended as follows: Plexy: 12-24=18cycles; 25-48=17 cycles; 48-96=16 cycles; 97-192=15 cycles; 193-384=14cycles; 385-768=13 cycles; 769-1536=12 cycles; 1537-3072=11 cycles. Whenusing fragmented DNA, up to 2 additional cycles were added to thepreamplification PCR process. Additionally, the annealing temperature(60° C.) can be increased from 4 minutes to 8 minutes when using 1537+plexy.

During the nick translation and library amplification step, the numberof PCR cycles can be increased, for example to about 10, when necessary.

The amplicon library was used to prepare DNA templates that wereclonally amplified on Ion Sphere™ Particles using emulsion PCR (emPCR).The preparation of template in the instant example was preparedaccording to the manufacturer's instructions using an Ion XpressTemplate Kit (Life Technologies, Part No. 4466457), hereby incorporatedby reference in its entirety. Once template-positive Ion SphereParticles were enriched, an aliquot of the Ion Spheres were loaded ontoan Ion 316™ Chip (Life Technologies, Part No. 4466616) as described inthe Ion Sequencing User Guide (Part No. 4467391), hereby incorporated inits entirety, and subjected to analysis and sequencing as described inthe Ion Torrent PGM Sequencer User Guide (Life Technologies, Part No.4462917), hereby incorporated in its entirety.

In this example, 10 ng FFPE DNA was applied as the input DNA. Theresults from this experiment are provided in the Table below anddemonstrated approximately 94% showed base without strand bias, greaterthan 98% per base accuracy, and 99% of bases with greater than 20×coverage when average coverage normalized to 100×.

Number of targets 1459 Per Base Accuracy 98.70% Percent bases >0.2x mean99.20% Target coverage at 20x if normalized to 99.20% 100x averagecoverage depth Base without Strand Bias 94.12% Percent of reads ontarget 80.54% Coverage needed for 98 percentile base 65.07 with at least20x coverage Percent bases >0.01x mean 99.77%

Example 19 Multiplex PCR with 12,500 Target Specific Primers

In this example, several amplicon libraries were prepared usingapproximately 12,000 target-specific primer pairs in a single reaction.The target-specific primer pairs were prepared from genes associatedwith cancers, provided in Table 18. The target-specific primers weredesigned using the primer selection criteria outlined herein. In thisexample, primers from Table 6 of U.S. Application 61/598,881 herebyincorporated by reference in its entirety were used as target-specificprimers in the reaction. Each target-specific primer pair was designedto promote amplification of the intended target sequence outlined inTable 18. Each amplicon library was prepared according to the section ofExample 13 titled PCR amplify genomic DNA targets, except that theconcentration of target-specific primers in the primer pool was amendedto 25 nM and the number of target-specific primers was about 12,000.Additionally, the preamplification PCR cycles was amended as follows: Aninitial holding stage was performed at 99° C. for 2 minutes, followed by11 cycles of denaturing at 99° C. for 15 seconds; a first annealingstage at 60° C. for 10 minutes; a second annealing stage at 63° C. for 5minutes; a third annealing stage at 66° C. for 5 minutes; a fourthannealing stage at 69° C. for 5 minutes; and an extending stage at 72°C. for 5 minutes; followed by 5 cycles of denaturing at 99° C. for 15seconds; a first annealing stage at 60° C. for 10 minutes; a secondannealing stage at 63° C. for 6 minutes; a third annealing stage at 66°C. for 6 minutes; a fourth annealing stage at 69° C. for 6 minutes; andan extending stage at 72° C. for 6 minutes. After cycling, thepreamplified amplicon library was held at 4° C. until proceeding to thenext step as outlined below.

Purify the Amplicons from Input DNA and Primers

One round of Agencourt® AMPure® XP Reagent (Beckman Coulter, CA)binding, wash, and elution at 1.2× volume ratio was found to removegenomic DNA and unbound or excess primers.

In a 1.5 ml LoBind tube (Eppendorf, Part No. 022431021), thepreamplified amplicon library (20 microliters) was combined with 24microliters (1.2× volume) of Agencourt® AMPure® XP reagent (BeckmanCoulter, CA). The bead suspension was pipetted up and down to thoroughlymix the bead suspension with the preamplified amplicon library. Thesample was then pulse-spin and incubated for 5 minutes at roomtemperature.

The tube containing the sample was placed on a magnetic rack such as aDynaMag™-2 spin magnet (Life Technologies, CA, Part No. 123-21D) for 2minutes to capture the beads. Once the solution cleared, the supernatantwas transferred to a new tube, where 13.3 ul of nuclease free water wasadded. Then, 48 microliters of AgenCourt® AMPure® XP beads (BeckmanCoulter, CA) were added to the diluted supernatant. The mixture waspipetted to ensure the bead suspension mixed with the preamplifiedamplicon library. The sample was then pulse-spin and incubated at roomtemperature for 5 minutes. The tube containing the sample was placed onthe magnetic rack for 2 minutes to capture the beads. Once the solutioncleared, the supernatant was carefully discarded without disturbing thebead pellet. The desired preamplified amplicon library was now bound tothe beads. Without removing the tube from the magnetic rack, 200microliters of freshly prepared 70% ethanol was introduced into thesample. The sample was incubated for 30 seconds while gently rotatingthe tube on the magnetic rack. After the solution cleared, thesupernatant was discarded without disturbing the pellet. A secondethanol wash was performed and the supernatant discarded. Any remainingethanol was removed by pulse-spinning the tube and carefully removingresidual ethanol while not disturbing the pellet. The pellet wasair-dried for about 5 minutes at room temperature.

Once the tube was dry, the tube was removed from the magnetic rack and20 microliters of DNase/RNase Free Water was added (Life Technologies,CA, Part No. 600004). The tube was vortexed and pipetted to ensure thesample was mixed thoroughly. The sample was pulse-spin and placed on themagnetic rack for two minutes. After the solution cleared, thesupernatant containing the eluted DNA was transferred to a new tube.

Phosphorylate the Amplicons

To the eluted DNA (˜20 microliters), 3 microliters of DNA ligase buffer(Invitrogen, Catalog No. 15224041), 2 microliters dNTP mix, and 2microliters of FuP reagent were added. The reaction mixture was mixedthoroughly to ensure uniformity and incubated at 37° C. for 13 minutes.

Ligate Adapters to the Amplicons and Purify the Ligated Amplicons

After incubation, the reaction mixture proceeded directly to a ligationstep. Here, the reaction mixture now containing the phosphorylatedamplicon library was combined with 1.5 microliter of A/P1 adapters (20μm each) (sold as a component of the Ion Fragment Library Kit, LifeTechnologies, Part No. 4466464) and 1 microliter of DNA ligase (sold asa component of the Ion Fragment Library Kit, Life Technologies, Part No.4466464), and incubated at room temperature for 30 minutes.

After the incubation step, 52 microliters (1.8× sample volume) ofAgenCourt® AMPure® Reagent (Beckman Coulter, CA) was added to theligated DNA. The mixture was pipetted thoroughly to mix the beadsuspension with the ligated DNA. The mixture was pulse-spin andincubated at room temperature for 5 minutes. The samples underwentanother pulse-spin and were placed on a magnetic rack such as aDynaMag™-2 spin magnet (Life Technologies, CA, Part No. 123-21D) for twominutes. After the solution had cleared, the supernatant was discarded.Without removing the tube from the magnetic rack, 200 microliters offreshly prepared 70% ethanol was introduced into the sample. The samplewas incubated for 30 seconds while gently rotating the tube on themagnetic rack. After the solution cleared, the supernatant was discardedwithout disturbing the pellet. A second ethanol wash was performed andthe supernatant discarded. Any remaining ethanol was removed bypulse-spinning the tube and carefully removing residual ethanol whilenot disturbing the pellet. The pellet was air-dried for about 5 minutesat room temperature.

The pellet was resuspended in 20 microliters of DNase/RNase Free Water(Life Technologies, CA, Part No. 600004) and vortexed to ensure thesample was mixed thoroughly. The sample was pulse-spin and placed on themagnetic rack for two minutes. After the solution cleared, thesupernatant containing the ligated DNA was transferred to a new Lobindtube (Eppendorf, Part No. 022431021).

Nick Translate and Amplify the Amplicon Library and Purify the Library

The ligated DNA (˜20 microliters) was combined with 76 microliters ofPlatinum® PCR SuperMix High Fidelity (Life Technologies, CA, Part No.12532-016, sold as a component of the Ion Fragment Library Kit, LifeTechnologies, Part No. 4466464) and 4 microliters of LibraryAmplification Primer Mix (5 μM each) (Life Technologies, CA, Part No.602-1068-01, sold as a component of the Ion Fragment Library Kit, LifeTechnologies, Part No. 4466464), the mixture was pipetted thoroughly toensure a uniformed solution. The solution was applied to a single wellof a 96-well PCR plate and sealed. The plate was loaded into a thermalcycler (GeneAmp® PCR system 9700 Dual 96-well thermal cycler (LifeTechnologies, CA, Part No. N8050200 and 4314445)) and run on thefollowing temperate profile to generate the final amplicon library.

A nick-translation was performed at 72° C. for 1 minute, followed by anenzyme activation stage at 98° C. for 2 minutes, followed by 6 cycles ofdenaturing at 98° C. for 15 seconds and an annealing and extending stageat 60° C. for 1 minute. After cycling, the final amplicon library washeld at 4° C. until proceeding to the final purification step outlinedbelow.

In a 1.5 ml LoBind tube (Eppendorf, Part No. 022431021), the finalamplicon library (˜100 microliters) was combined with 180 microliters(1.8× sample volume) of Agencourt® AMPure® XP reagent (Beckman Coulter,CA). The bead suspension was pipetted up and down to thoroughly mix thebead suspension with the final amplicon library. The sample was thenpulse-spin and incubated for 5 minutes at room temperature.

The tube containing the final amplicon library was placed on a magneticrack such as a DynaMag™-2 spin magnet (Life Technologies, CA, Part No.123-21D) for 2 minutes to capture the beads. Once the solution cleared,the supernatant was carefully discarded without disturbing the beadpellet. Without removing the tube from the magnetic rack, 400microliters of freshly prepared 70% ethanol was introduced into thesample. The sample was incubated for 30 seconds while gently rotatingthe tube on the magnetic rack. After the solution cleared, thesupernatant was discarded without disturbing the pellet. A secondethanol wash was performed and the supernatant discarded. Any remainingethanol was removed by pulse-spinning the tube and carefully removingresidual ethanol while not disturbing the pellet. The pellet wasair-dried for about 5 minutes at room temperature.

Once the tube was dry, the tube was removed from the magnetic rack and20 microliters of Low TE was added (Life Technologies, CA, Part No.602-1066-01). The tube was pipetted and vortexed to ensure the samplewas mixed thoroughly. The sample was pulse-spin and placed on themagnetic rack for two minutes. After the solution cleared, thesupernatant containing the final amplicon library was transferred to anew Lobind tube (Eppendorf, Part No. 022431021).

Assess the Library Size Distribution and Determine the Template DilutionFactor

The final amplicon library was quantitated to determine the librarydilution (Template Dilution Factor) that results in a concentrationwithin the optimized target range for Template Preparation (e.g.,PCR-mediated addition of library molecules onto Ion Sphere™ Particles).The final amplicon library is typically quantitated for downstreamTemplate Preparation procedure using an Ion Library Quantitation Kit(qPCR) (Life Technologies, Part No. 4468802) and/or a Bioanalyzer™(Agilent Technologies, Agilent 2100 Bioanalyzer) to determine the molarconcentration of the amplicon library, from which the Template DilutionFactor is calculated. For example, instructions to determine theTemplate Dilution Factor by quantitative real-time PCR (qPCR) can befound in the Ion Library Quantitation Kit User Guide (Life Technologies,Part No. 4468986), hereby incorporated by reference in its entirety.

In this example, 1 microliter of the final amplicon library preparationwas analyzed on the 2100 Bioanalyzer™ with an Agilent High SensitivityDNA Kit (Agilent Technologies, Part No. 5067-4626) to generate peaks inthe 135-205 bp size range and at a concentration of about 5×10⁹ copiesper microliter.

Proceed to Template Preparation

An aliquot of the final library was used to prepare DNA templates thatwere clonally amplified on Ion Sphere™ Particles using emulsion PCR(emPCR). The preparation of template in the instant example was preparedaccording to the manufacturer's instructions using an Ion XpressTemplate Kit (Life Technologies, Part No. 4466457), hereby incorporatedby reference in its entirety. Once template-positive Ion SphereParticles were enriched, an aliquot of the Ion Spheres were loaded ontoan Ion 314™ Chip (Life Technologies, Part No. 4462923) as described inthe Ion Sequencing User Guide (Part No. 4467391), hereby incorporated inits entirety, and subjected to analysis and sequencing as described inthe Ion Torrent PGM Sequencer User Guide (Life Technologies, Part No.4462917), hereby incorporated in its entirety. The data obtained fromthis example is provided in the Table below.

Ampliseq 1.0 work flow mod Sample (50 ng NA12878) enrich unbound Plexity6,000 12,000 12,000 12,000 C450 oligo pool pool 1 + 3 pool 1 + 2 + 3 + 4pool 1 + 2 + 3 + 4 pool 1 + 2 + 3 + 4 PreAmp cycle (6 hr = PA1, 9 hr =PA2) 6 hr 6 hr 9 hr 9 hr PA enzyme/load % Taq/69% Taq/82% Stoffel/15%Stoffel/43% Percent greater than 0.01 mean reads per base 97.72% 79.15%82.08% 78.42% Percent greater than 0.1 mean reads per base 84.32% 54.28%82.08% 54.22% Percent greater than 0.2 mean reads per base 73.79% 44.86%68.80% 45.45% Percent no strand bias of all bases 80.42% 68.48% 45.19%66.01% Percent no strand bias of all Amps 81.23% 62.87% 32.58% 54.80%Percent no strand bias of all Amps >100 reads 139.07% 229.75% 8126.00%334.62% Percent end to end read of on target reads 71.40% 69.05% 53.32%77.87% Per base accuracy 97.38% 97.47% 97.18% 98.87% Percent of totalreads mapped to hg19 98.17% 97.86% 97.85% 97.10% Percent wells with read25.93% 30.24% 1.62% 12.97% Number of total reads 1643486 1916570 102466821799 Number of mapped reads 1613458 1875619 100261 797953 Number oftargets 6249 12469 12469 12469 Number of reads on target 1534973 179383593560 763982 Percent all reads on target 93.40% 93.60% 91.31% 92.96%Percent mapped reads on target 95.14% 95.64% 93.32% 95.74% Percent readsoff target 4.78% 4.27% 6.54% 4.13% Percent reads unmapped 1.83% 2.14%2.15% 2.90% Bases in targeted reference 668162 1249178 1249178 1249178Bases covered (at least 1x) 661416 1076079 1025284 979564 Total basereads on target 156995237 189835759 9789500 80845870 Average basecoverage depth 234.97 151.97 7.84 64.72 Maximum base read depth 23914284 339 3921 Average base read depth 237.32 176.23 9.52 82.46 Std. Devbase read depth 277.21 351.91 16.09 157.73 Target coverage at 1x 98.99%86.14% 82.08% 78.42% Target coverage at 10x 93.38% 60.43% 22.25% 49.45%Target coverage at 20x 86.73% 51.08% 9.49% 39.12% Target coverage at100x 57.38% 28.52% 0.44% 17.75% Target coverage at 500x 14.24% 9.07%0.00% 2.34% Target coverage at 1x - norm 100 97.72% 79.15% 82.08% 78.42%Target coverage at 10x - norm 100 84.32% 54.28% 82.08% 54.22% Targetcoverage at 20x - norm 100 73.79% 44.86% 68.80% 45.45% Target coverageat 100x - norm 100 33.67% 23.15% 27.66% 23.26% Target coverage at 500x -norm 100 1.20% 5.22% 3.24% 5.22% Percent end to end read of on targetreads 71.40% 69.05% 53.32% 77.87% Percent forward end to end read of ontarget reads 36.45% 35.07% 26.64% 39.40% Percent reverse end to end readof on target reads 34.95% 33.98% 26.67% 38.47% Coverage needed for 99percentile base with at least 156.65 475.51 215.63 274.86 1x coverage

Example 20 Assessment of Relative Copy Number Variation (CNV)

In this example, several DNA samples were amplified using 3000target-specific primer pairs. The target-specific primers were designedusing the target-specific primer selection criteria outlined herein. Thesequences of the selected target-specific primers used in thisexperiment can be found in Table 17 (or Table 6 of U.S. Application61/598,881 hereby incorporated by reference in its entirety). The DNAsamples were barcoded during the library amplification process (asoutlined in Example 13). In this example, the DNA samples were obtainedfrom a commercial source (Coriell DNA) and contained known variations incopy number to demonstrate that the multiplex amplification methodsdisclosed herein can be used to assess copy number variation.

Four DNA samples were purchased from Coriell DNA that contained a 3 Mbdeletion on chromosome 22 that was associated with DiGeorge Syndrome. Anadditional DNA sample was purchased from Coriell DNA that contained a 16Mb deletion on chromosome 7 associated with Grieg CephalopolysyndactylySyndrome (GCPS). An amplicon library was prepared for each DNA sampleincluding a barcode for the purposes of distinguishing one DNA samplefrom another. Each amplicon library was prepared according to the methodoutlined in Example 15, except in this example, 3000 target-specificprimer pairs were used in a single preamplification reaction. Thetarget-specific primer pairs were prepared from the genes in Table 18.Each amplicon library was prepared using 10 ng of DNA from the startingmaterial. The libraries were prepared according to Example 15 exceptthat the number of preamplification PCR cycles was amended as follows:Plexy: 12-24=18 cycles; 25-48=17 cycles; 48-96=16 cycles; 97-192=15cycles; 193-384=14 cycles; 385-768=13 cycles; 769-1536=12 cycles;1537-3072=11 cycles. When using fragmented DNA (e.g., FFPE DNA samples),up to 2 additional cycles can be added to the preamplification PCRprocess. Additionally, the annealing temperature (60° C.) can beincreased from 4 minutes to 8 minutes when using 1537+ plexy, if ahigher yield is necessary.

If required, the number of PCR cycles can be increased during the nicktranslation and library amplification step, for example to about 10cycles.

The amplicon library was used to prepare DNA templates that wereclonally amplified on Ion Sphere™ Particles using emulsion PCR (emPCR).The preparation of template in the instant example was preparedaccording to the manufacturer's instructions using an Ion XpressTemplate Kit (Life Technologies, Part No. 4466457), hereby incorporatedby reference in its entirety. One template-positive Ion Sphere Particleswere enriched, an aliquot of the Ion Spheres were loaded onto an Ion316™ Chip (Life Technologies, Part No. 4466616) as described in the IonSequencing User Guide (Part No. 4467391), hereby incorporated in itsentirety, and subjected to analysis and sequencing as described in theIon Torrent PGM Sequencer User Guide (Life Technologies, Part No.4462917), hereby incorporated in its entirety.

In this example, 10 ng DNA was applied as the input DNA. The resultsfrom this experiment are provided in FIGS. 14-15.

FIG. 14 shows that the multiplex PCR amplification methods disclosedherein can be used to assess copy number variation. FIG. 14 shows theamplicon frequency data for 12 amplicons spanning part of the criticalregion of the gene CLTCL1 associated with DiGeorge Syndrome. The circlesplotted at frequency 0 represent each amplicon of CLTCL1 amplified bythe multiplex amplification method. The red (solo) circles are datapoints from a single DiGeorge Syndrome DNA sample obtained from CoriellDNA. Four DiGeorge Syndrome DNA samples were tested in this experimentand an average ratio of percent total reads filtered for DiGeorgesamples compared to controls are plotted as circles with error bars. Thedotted line at frequency 1.0 is the frequency expected for a normalsample (i.e., a sample not containing a variation in copy number). As isshown in FIG. 14, the frequencies of the tested DiGeorge Syndromesamples are about 0.5, indicating that a deletion within the CLTCL1 genehas occurred and can be observed and detected using the amplificationmethods disclosed herein.

FIG. 15 shows that the multiplex amplification methods disclosed hereincan be used to assess copy number variation in a different chromosome.FIG. 15 shows the amplicon frequency data for 4 amplicons spanning partof the gene IKZF1, associated with Greig Cephalopolysyndactyly Syndrome(GCPS). The circles plotted at frequency 0 represent each of the fouramplicons of IKZF1 that were amplified by the multiplex PCRamplification method outlined in this example. Ratios were obtained fora single GCPS sample as compared to four control samples and theirfrequencies are plotted in FIG. 15. GCPS DNA has only one copy of theGCPS gene, whereas normal (or control) DNA samples contain two copies ofthe gene. As noted in FIG. 15, the control samples show 2× for copynumber as compared to the expected frequency (dotted line). As isdemonstrated by FIG. 15, variation in copy number can be determinedusing the multiplex PCR amplification method disclosed herein.

Example 21 Mutiplex Amplification with an Inosine Cleavable Group

In this example, target-specific primers containing one or morecleavable groups denoted as an inosine were prepared according to theprimer selection criteria disclosed herein. After an initial in silicoevaluation of the primer pairs to proposed target sequences, theevaluated target-specific primers (disclosed in Table 19) were orderedfrom Integrated DNA Technologies (IDT) (Coravilla, Iowa). The inosinecontaining primers were received from IDT and subjected to multiplexamplification, performed according to the method of Example 15 with thefollowing exceptions. The 2× Primer Pool (containing primer from Table2) was replaced with the Inosine containing primers (disclosed on Table19, corresponding to SEQ ID NOs: 103122-103143). Additionally, afterperforming PCR amplification of genomic DNA targets, the samples wassubjected to digestion with EndoV and FuPa.

In this example, an aliquot of the final library was used to prepare DNAtemplates that were clonally amplified on Ion Sphere™ Particles usingemulsion PCR (emPCR). The preparation of template in the instant examplewas prepared according to the manufacturer's instructions using an IonXpress Template Kit (Life Technologies, Part No. 4466457), herebyincorporated by reference in its entirety. Once template-positive IonSphere Particles were enriched, an aliquot of the Ion Spheres wereloaded onto an Ion 314™ Chip (Life Technologies, Part No. 4462923) asdescribed in the Ion Sequencing User Guide (Part No. 4467391), herebyincorporated in its entirety, and subjected to analysis and sequencingas described in the Ion Torrent PGM Sequencer User Guide (LifeTechnologies, Part No. 4462917), hereby incorporated in its entirety.The data obtained from this example included a per base accuracypercentage of 97.17% and percent of reads on target were observed as96.83%.

Example 22 Alternative Multiplex PCR Protocol

In this example, various alternative embodiments are presented over oneor more of the prior multiplex PCR methods.

PCR Amplify Genomic DNA Targets

In this example, a multiplex polymerase chain reaction was performed toamplify multiple individual amplicons (target sequences) across agenomic DNA sample and an FFPE sample. A representative list of genesassociated with cancers and inherited diseases that were incorporatedfor investigation while synthesizing the primer pool is provided in atleast Tables 1 and 4. Each primer pair in the primer pool was designedto contain at least one uracil nucleotide in each of the forward andreverse primer (Table 2) or designed to contain at least one inosineresidue in each of the forward and reverse primer (Table 19). Eachprimer pair was also designed to selectively hybridize to, and promoteamplification of a specific gene or gene fragment of the genomic DNA orFFPE sample to reduce formation of non-specific amplification products.

To a single well of a 96-well PCR plate was added 10 microliters of 2×Primer Pool (containing the primer pairs of Table 2), 4 microliters of5× Cancer Primer pool (containing the primer pairs of Table 17), 4microliters of 5×IDP Primer pool (containing the primer pairs of Table15) or 10 microliters of 2×HID Primer Pool (containing the primer pairsof Tables 13 and 14), at a concentration of 200 nm each for plexy under96, or at a concentration of 50 nm for plexy above 96. 10 ng of genomicDNA or FFPE DNA and 4 microliters of an amplification reaction mixture(5× Ion Ampliseq HiFi Master Mix) was added to a final volume of 20microliters with nuclease free water (Life Technologies, CA, Part No.600004).

The PCR plate was sealed and loaded into a thermal cycler (GeneAmp® PCRsystem 9700 Dual 96-well thermal cycler (Life Technologies, CA, Part No.N8050200 and 4314445)) and run on the following temperature profile togenerate the preamplified amplicon library. Variation to the number ofcycles was performed based on the total plexy of the reaction mixtureunder investigation. For example, a plexy of 12-24 was run for 20cycles; a plexy of 25-48 was run for 19 cycles; a plexy of 48-96 was runfor 18 cycles; a plexy of 97-192 was run for 17 cycles; a plexy of193-384 was run for 16 cycles; a plexy of 385-768 was run for 15 cycles;a plexy of 769-1536 was run for 14 cycles; a plexy of 1537-3072 was runfor 13 cycles; a plexy of 3073-6144 was run for 12 cycles. Additionally,for reaction mixtures containing barcodes or pooled reaction mixturesthe number of cycles was lowered by one or more additional cycles. Forexample, 2-3 barcodes per sample were subtracted by one cycle; 4-8barcodes per sample were subtracted by 2 cycles, and 9-16 barcodes weresubtracted by 3 cycles. For samples that contain fragmented DNA, e.g.,FFPE or enzymatically digested DNA, the number of cycles can beincreased for up to 3 cycles.

An initial holding stage was performed at 99° C. for 2 minutes, followedby X cycles (as determined above) of denaturing at 99° C. for 15 secondsand an annealing and extending stage at 60° C. for 4 minutes. For plexyabove 1536, the annealing and extending stage was increase to 8 minutesat 60° C. After cycling, the preamplified amplicon library was held at10° C. until proceeding to the purification step outlined below.

Digest/Phosphorylate/Heat Kill the Amplicons

To the preamplified library (˜20 microliters), 2 microliters of FuPareagent was added. The PCR plate was sealed and loaded into a thermalcycler (GeneAmp® PCR system 9700 Dual 96-well thermal cycler (LifeTechnologies, CA, Part No. N8050200 and 4314445)) and run on thefollowing temperature profile. An initial holding stage was performed at50° C. for 10 minutes, followed by 55° C. for 10 minutes and then 65° C.for 20 minutes. After cycling, the preamplified amplicon library washeld at 10° C. until proceeding to the ligation/nick translation stepoutlined below.

Ligate Adapters to the Amplicons and Nick Translate

After phosphorylation, the amplicon preamplification library (˜22 μl)proceeded directly to a ligation step. In this example, thepreamplification library now containing the phosphorylated ampliconlibrary was combined with 2 microliters of A/P1 Adapter (5 μm each)(sold as a component of the Ion Fragment Library Kit, Life Technologies,Part No. 4466464), 4 microliters of Switch Solution and 2 microliters ofDNA ligase (5 u/μl). The PCR plate was sealed and loaded into a thermalcycler (GeneAmp® PCR system 9700 Dual 96-well thermal cycler (LifeTechnologies, CA, Part No. N8050200 and 4314445)) and run on thefollowing temperature profile. An initial holding stage was performed at22° C. for 30 minutes, followed by 72° C. for 10 minutes and then heldat 10° C. until proceeding to the next step.

If the amplicon library is to contain barcodes (for example Ion DNABarcoding 1-16 kit, Life Technologies, Part No. 4468654, incorporatedherein in its entirety), the barcodes are added at this step to the PCRplate essentially according to the manufacturer's instructions prior toproceeding to the next step. Optionally, all the samples or barcodes canbe pooled into a single tube at this step.

1.5× AMPure XP Purification

1.5× sample volume (45 microliters) of AgenCourt® AMPure® Reagent(Beckman Coulter, CA) was added to the ligated DNA. The mixture wasmixed and incubated at room temperature for 5 minutes and thentransferred to a magnet plate. Sample was incubated on the magnet platefor 2 minutes, and the supernatant discarded. An ethanol wash wasperformed and the supernatant discarded. Any remaining ethanol wasremoved and air-dried for about 5 minutes at room temperature. The drytube containing the library was resuspended in 20 microliters ofNuclease Free Water (Life Technologies, CA, Part No. 600004) or lowTris-EDTA buffer. In some instances, an optional nicktranslation/library amplification step can be performed on the ampliconlibrary, as outlined below.

Nick Translate and Amplify the Amplicon Library and Purify the Library

The ligated DNA (˜20 microliters) was combined with 50 microliters ofPlatinum® PCR SuperMix High Fidelity (Life Technologies, CA, Part No.12532-016, sold as a component of the Ion Fragment Library Kit, LifeTechnologies, Part No. 4466464) and placed on a magnet plate for 2minutes. 48 ul of the eluted amplicons in the PCR supermix weretransferred to a new well of a 96-well plate to which was added 2microliters of Library Amplification Primer Mix (Life Technologies, CA,Part No. 602-1068-01, sold as a component of the Ion Fragment LibraryKit, Life Technologies, Part No. 4466464). The PCR plate was sealed andloaded into a thermal cycler (GeneAmp® PCR system 9700 Dual 96-wellthermal cycler (Life Technologies, CA, Part No. N8050200 and 4314445))and run on the following temperate profile to generate the finalamplicon library.

An enzyme activation stage at 98° C. for 2 minutes, followed by 5 cyclesof denaturing at 98° C. for 15 seconds and an annealing and extendingstage at 60° C. for 1 minute. After cycling, the final amplicon librarywas held at 4° C. until proceeding to the final purification stepoutlined below.

The final amplicon library (˜100 microliters) was combined with 0.5×sample volume of Agencourt® AMPure® XP reagent (Beckman Coulter, CA).The mixture was then incubated for 5 minutes at room temperature. Thesample was transferred to a magnet plate for 2 minutes and thesupernatant (˜75 microliters) removed to a new well. 1.2× volume ofAgencourt® AMPure® XP reagent (Beckman Coulter, CA) was added andincubated at room temperature for 5 minutes. The sample was then placedon a magnet plate for 2 minutes and the supernatant discarded. The finalamplicon library was washed with 70% ethanol and the supernatantdiscarded. Any remaining ethanol was removed and air-dried for about 5minutes at room temperature. Once dry, the library was resuspended in 20microliters of Low TE (Life Technologies, CA, Part No. 602-1066-01). Inthis example, 1 microliter of the final amplicon library preparation wasanalyzed on a 2100 Bioanalyzer™ with an Agilent High Sensitivity DNA Kit(Agilent Technologies, Part No. 5067-4626) or analyzed on a Qubitmachine using the DSDNA HS Assay Kit (Part number Q32851).

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LENGTHY TABLES The patent application contains a lengthy table section.A copy of the table is available in electronic form from the USPTO website(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20120295819A1).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

1. A method for amplifying a plurality of different target sequenceswithin a sample, comprising: amplifying within a single amplificationreaction mixture at least one hundred different target sequences from asample including a plurality of different target sequences, wherein theamplifying includes contacting at least some portion of the sample witha plurality of target-specific primers, and a polymerase underamplification conditions, thereby producing at least one hundreddifferent amplified target sequences, wherein at least two of thedifferent amplified target sequences are less than 50% complementary toeach other and wherein at least one of the plurality of target-specificprimers and at least one of the amplified target sequences includes acleavable group; cleaving a cleavable group of at least one amplifiedtarget sequence; ligating at least one adapter to at least one amplifiedtarget sequence in a blunt-ended ligation reaction, thereby producingone or more adapter-ligated amplified target sequences, and reamplifyingat least one of the adapter-ligated amplified target sequences.
 2. Themethod of claim 1, wherein one or more of the at least one adapter isnot substantially complementary to at least one amplified targetsequence.
 3. The method of claim 1, wherein the reamplifying includescontacting the at least one adapter-ligated amplified target sequencewith one or more primers including a sequence that is complementary toat least one of the adapters or their complement, and a polymerase underamplification conditions, thereby producing at least one reamplifiedadapter-ligated amplified target sequence.
 4. The method of claim 3,wherein at least one of the one or more adapters or their complements isnot substantially complementary to at least one amplified targetsequence.
 5. The method of claim 1, wherein at least one target-specificprimer is substantially complementary to at least a portion of acorresponding target sequence in the sample.
 6. The method of claim 1,wherein an adapter that is ligated to at least one of the amplifiedtarget sequences is susceptible to exonuclease digestion.
 7. The methodof claim 1, wherein an adapter that is ligated to at least one of theamplified target sequences does not include a protecting group.
 8. Themethod of claim 1, wherein the ligating includes contacting at least oneamplified target sequence having a 3′ end and a 5′ end with a ligationreaction mixture including one or more adapters and a ligase underligation conditions, wherein none of the adapters in the ligationreaction mixture includes, prior to the ligating, a target-specificsequence.
 9. The method of claim 8, wherein the ligating includescontacting at least one amplified target sequence with a ligationreaction mixture including one or more adapters and a ligase underligation conditions, wherein the ligation reaction mixture does notinclude one or more additional oligonucleotide adapters prior toligating the one or more adapters to at least one amplified targetsequence.
 10. The method of claim 1, wherein the amplifying furtherincludes a digestion step prior to the ligating, thereby producing aplurality of blunt-end amplified target sequences possessing a 5′phosphate group.